Title STOP1 転写制御による植物の酸性土壌耐性機構に関する研究( 本文(Fulltext) ) Author(s) 澤木, 宣忠 Report No.(Doctoral Degree) 博士(農学) 乙第141号 Issue Date 2014-03-13 Type 博士論文 Version ETD URL http://hdl.handle.net/20.500.12099/49026 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。
STOP1 ㌿ไᚚࡼࡿ
᳜≀ࡢ㓟ᛶᅵተ⪏ᛶᶵᵓ㛵ࡍࡿ◊✲
2013 ᖺ
ᒱ㜧ᏛᏛ㝔㐃ྜ㎰Ꮫ◊✲⛉
STOP1 ㌿ไᚚࡼࡿ
᳜≀ࡢ㓟ᛶᅵተ⪏ᛶᶵᵓ㛵ࡍࡿ◊✲
2
┠
┠ḟ
ᗎㄽ
➨
1 ❶ ࢩࣟࢾࢼࢬࢼࡢ Alࠊప pH ⪏ᛶ㌿ᅉᏊ
AtSTOP1 ࡀไᚚࡍࡿୗὶ㑇ఏᏊࡢ᥈⣴
➨
2 ❶ ࣮ࣘ࢝ࣜࡢ Al ⪏ᛶ㑇ఏᏊ AtSTOP1
࣍ࣔࣟࢢ㑇ఏᏊࡢ㛵㐃ᛶ
⤖ㄽ
せ⣙
ㅰ㎡
ᘬ⏝ᩥ⊩
3 㡫
9 㡫
52 㡫
93 㡫
96 㡫
99 㡫
100 㡫
3
ᗎ
ᗎㄽ
᪥ࠊୡ⏺ࡢேཱྀࡣ70 ൨ேࢆ㉸࠼(2011 ᖺ)ࠊ2050 ᖺࡲ࡛ࡣ 90 ൨ேࢆ㉸࠼
ࡿぢ㎸ࡲࢀࡿࠋࡋࡋ୍᪉࡛ࡣ㣚㣹ࡀ῝้ࡋࠊᰤ㣴㊊ࡢேࡣୡ⏺࠾࠸࡚
⣙9 ൨ே࠸ࡿ᥎ᐃࡉࢀ࡚࠸ࡿ [FOOD AND AGRICULTURE ORGANIZATION
OF THE UNITED NATIONS (FAO)]ࠋࡑࡢࡓࡵ㣗⣊㊊ࡢゎᾘࡀᛴົ࡞ࡗ࡚࠸ ࡿࡀࠊୡ⏺ⓗస≀ࡢ⏕⏘⬟ຊࡣ㝈⏺࡞ࡗ࡚࠸ࡿࡢࡀ⌧≧࡛࠶ࡿࠋࡦࡘࡢ⌮⏤ ࡋ࡚ࡣࠊࡇࢀࡲ࡛ࡢရ✀ᨵⰋࡢ⤖ᯝࠊ⏕⏘ᛶࡸရ㉁ࡀ༑ศ㧗ࡃ࡞ࡗࡓࡓࡵࡇ ࢀ௨ୖࡢⓗ࡞ᨵⰋࡀᮇᚅ࡛ࡁ࡞ࡃ࡞ࡗ࡚࠸ࡿࡇࡀ࠶ࡿࠋࡑࡋ࡚ࡶ࠺ࡦࡘࡣస ≀ࡢ⪔ᆅ㠃✚ࡢᣑࡀᅔ㞴࡞ࡗ࡚࠸ࡿࡇ࡛࠶ࡿࠋࡇࢀࡣࡦ࠼ࠊస≀⏕⏘ 㐺ࡉ࡞࠸ⰋᅵተࡀᗈࡃᏑᅾࡋ࡚࠸ࡿࡓࡵ࡛࠶ࡿࠋ୍⯡ࡇࡢࡼ࠺࡞ᅵተ࠾࠸࡚ ࡣస≀⏕⏘⬟ຊࡀⴭࡋࡃపୗࡍࡿࠋࡶࡋࠊࡇࡢࡼ࠺࡞ᅵተ࡛స≀⏕⏘ࢆ⾜࠸ࠊ✭ 㔞ࢆྥୖࡉࡏࡿࡇࡀ࡛ࡁࢀࡤࠊ㣗⣊㊊ࡢゎᾘྥࡅࡓ➨୍Ṍࡀ㋃ࡳฟࡏࡿ⪃ ࠼ࡽࢀࡿࠋ࡛ࡣࠊⰋᅵተࡣࡢࡼ࠺࡞✀㢮ࡀ࠶ࡾࠊఱࡀཎᅉ࡞ࡗ࡚⏕⫱Ⰻࢆ ᘬࡁ㉳ࡇࡋ࡚࠸ࡿࡢ࡛࠶ࢁ࠺ࠋࡲࡓࠊⰋᅵተ࡛స≀ࢆ⏕⏘ࡍࡿࡓࡵࡣ࠺ࡍ ࢀࡤࡼ࠸ࡢ࡛࠶ࢁ࠺ࠋ ୡ⏺ࡢ㝣ᆅ㠃✚ࡣ⣙130 ൨࣊ࢡࢱ࣮ࣝゝࢃࢀ࡚࠸ࡿࡀࠊ㎰⏝ᆅࡋ࡚⏝ࡉ
ࢀ࡚࠸ࡿࡢࡣ⣙15.5 ൨࣊ࢡࢱ࣮࡛ࣝ࠶ࡿ (FAO, FAO Statistical Yearbook 2012)ࠋ
ࡋࡋࠊ㎰⏝ᆅࡢ࠺ࡕࠊ㓟ᛶᅵተࡸࣝ࢝ࣜᛶᅵተࠊ⇱ᆅࡸ㔜㔠ᒓởᰁᅵተ࡞ ⣙70 㸣ࡀⰋᅵተゝࢃࢀ࡚࠸ࡿࠋࡇࡢ୰࡛≉ὀ┠ࡍࡁࡶࡢࡣࠊⰋᅵተࡢ ࠺ࡕ⣙50 㸣 (⣙ 5 ൨࣊ࢡࢱ࣮ࣝ)ࢆ༨ࡵ࡚࠸ࡿ㓟ᛶᅵተ࡛࠶ࡿࠋ㓟ᛶᅵተࡣୡ⏺୰ ᗈࡀࡗ࡚࠸ࡿࡀࠊ≉⇕ᖏࡸ ᖏ࡞㞵ࡢከ࠸ᆅᇦศᕸࡋ࡚࠸ࡿࠋࡇࢀࡣ✵Ẽ ୰ࡢ CO2ࡀ㞵Ỉ⁐ࡅࠊᅵተ୰࡛ᙅ㓟ᛶࢆ♧ࡍࡼ࠺࡞ࡿࡓࡵ࡛࠶ࡿࠋࡉࡽ㏆ ᖺ࡛ࡣࠊ⏘ᴗάືࡼࡿ❅⣲㓟≀ࡸ◲㯤㓟≀ࡀ㞵Ỉ⁐ࡅ㎸ࡴ㓟ᛶ㞵ࡢᙳ㡪ࠊ
4 ❅⣲ࢆከࡃྵࡴ⫧ᩱࡢᢞࡼࡿ◪㓟ែࡢቑຍࡢࡓࡵࠊᅵተࡢ㓟ᛶࡣ᪥ࠎ㐍⾜ࡋ ࡚࠸ࡿࡶゝࢃࢀ࡚࠸ࡿࠋ ࡇࡢࡼ࠺࡞⫼ᬒࡽࠊ㓟ᛶᅵተ࡛స≀⏕⏘࡛ࡁࡿࡼ࠺࡞ࡿࡇࡣ㠀ᖖព⩏ ࡀࡁ࠸⪃࠼ࡽࢀࠊࡇࢀࢆᐇ⾜ࡍࡿࡓࡵࡣ2 ࡘࡢ᪉ἲࡀ࠶ࡿࠋࡦࡘࡣᅵተ⮬ యࢆᨵⰋࡍࡿࡇࠊࡍ࡞ࢃࡕᅵተࡢ୰࡛࠶ࡿࠋࡋࡋࠊࡇࡢ᪉ἲ࡛ࡣከ㔞ࡢ୰ ⏝▼⅊ࡸ⫧ᩱࡀᚲせ࡞ࡿୖࠊ㓟ᛶᅵተࡀⓎᒎ㏵ୖᅜከࡃศᕸࡋ࡚࠸ࡿࡇࡽ ⤒῭ⓗࡶ⌧ᐇⓗ࡛ࡣ࡞࠸ࠋࡑࡇ࡛ࡶ࠺ࡦࡘࡢ᪉ἲࡋ࡚ࠊ᳜≀ࡢᨵⰋࠊࡍ࡞ࢃ ࡕ᳜≀ࡢ㓟ᛶᅵተ⪏ᛶࡢࡀᣲࡆࡽࢀࡿࠋࡇࢀࢆ⾜࠺ࡓࡵࡣࠊࡲࡎ㓟ᛶᅵተ ࡀ᳜≀ᑐࡋࡢࡼ࠺࡞ᙳ㡪ࢆ࠼࡚࠸ࡿࡢࢆ⌮ゎࡍࡿᚲせࡀ࠶ࡿࠋ㓟ᛶᅵተ ࡘ࠸࡚ࡢ◊✲ࡣ 1960 ᖺ௦ࡽ⌧ᅾࡲ࡛ᖜᗈࡃ⾜ࢃࢀࠊࡇࢀࡲ࡛࣑ࣝࢽ࣒࢘
(Al)ẘᛶࠊ࣐ࣥ࢞ࣥ (Mn)ẘᛶࠊ㕲 (Fe)ẘᛶࠊࣜࣥ㓟Ḟஈࠊ࢝ࣝࢩ࣒࢘ (Ca)Ḟஈࠊ
࣐ࣥ࢞ࣥ (Mn)Ḟஈࠊ࣒࢝ࣜ࢘ (K)Ḟஈࡀ」ྜⓗ㉳ࡁ࡚࠸ࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ
(von Uexküll and Mutert, 1995)ࠋලయⓗࡣࠊప pH ࡼࡗ࡚ྍ⁐ࡉࢀࡓᅵተ
୰ࡢ㔠ᒓ࢜ࣥ (Al3+ ࡞)ࠊ࠶ࡿ࠸ࡣỈ⣲࢜ࣥ (H+)ࡀ᳜≀┤᥋ᐖࢆ࠼࡚࠸ ࡿࡇࡀ࠶ࡿࠋࡉࡽ㛫᥋ⓗ࡞ᐖࡋ࡚ࠊࡑࡢྍ⁐ࡋࡓ㔠ᒓ࢜ࣥࣜࣥ㓟ࡀ⤖ ྜࡋ⁐ࡍࡿࡇ࡛᳜≀ࡣࣜࣥ㓟ࢆ྾࡛ࡁ࡞ࡃ࡞ࡿᰤ㣴Ḟஈࠊຍ࠼࡚ᅵተ୰ࡢ Ca ࡸ Mg ࡞ࡢሷᇶࡀ H+࢜ࣥࡉࢀὶฟࡍࡿࡇ࡛㉳ࡇࡿᰤ㣴Ḟஈࡶྠ Ꮡᅾࡍࡿࠋࡇࡢ୰࡛ࡶAl ࢜ࣥࡘ࠸࡚ࡣ≉ẘᛶࡀᙉࡃࠊ㓟ᛶᅵተ࠾ࡅࡿ స≀⏕⫱ࡢไ㝈せᅉࡢࡦࡘ࡞ࡗ࡚࠾ࡾࠊ⢭ຊⓗ◊✲ࡀ㐍ࡵࡽࢀ࡚࠸ࡿࠋ Al ࡣᅵተ୰᭱ࡶከࡃᏑᅾࡋ࡚࠸ࡿ㔠ᒓඖ⣲࡛࠶ࡿࠋpH ࡀ୰ᛶࡢሙྜࠊAl ࡣ㖔≀୰ࡢࢣ㓟⤖ྜࡋ࡚⁐ࡋ࡚࠸ࡿࡀࠊpH ࡢపୗక࠸ Al ࡀྍ⁐ࡋࠊ ᛶ㝧࢜ࣥࡋ࡚ᅵተ⢏Ꮚ྾╔ࡉࢀࡿࠋpH 5 ௨ୗ࡛ࡣࡇࢀࡀ⁐ฟࡋࠊẘᛶ ࡢᙉ࠸Al3+ ࡞ࡗ࡚᳜≀ᐖࢆ࠼ࡿࠋ㒊ศࡢ Al ࡣ᰿➃ࡢ⾲⓶㞟✚ࡋࠊࡲࡓ ࠶ࡿ୍㒊ࡣ⣽⬊ෆࡲ࡛ධࡍࡿࡇ࡛㞀ᐖࢆᘬࡁ㉳ࡇࡍࠋࡇࡢࡓࡵࠊAl ࢜ࣥ
5 ࡼࡿ㞀ᐖࡀ㢧ⴭ⾲ࢀࡿࡢࡣ᰿ࡢఙ㛗㜼ᐖ࡛࠶ࡾࠊ᰿➃ࡢศᇦ࠶ࡿ⣽⬊ࡢఙ 㛗ࡀ㜼ᐖࡉࢀࡿࠋࡉࡽഃ᰿ࡢⓎ⏕ࠊ〓Ⰽࠊ᰿ࡀኴࡃ࡞ࡿ➼ࡢ≧ࡶ⾲ࢀࡿࡇ ࡀ▱ࡽࢀ࡚࠸ࡿࠋ୍᪉ࠊศᏊ⏕≀࡛ࣞ࣋ࣝࡢࡇࢀࡽࡢ㞀ᐖᛂ⟅ᶵᵓࡣᮍゎ᫂ࡢ㒊ศ ࡀከ࠸ࡀࠊAl ࡼࡿ⣽⬊ෆࡢኚࡘ࠸࡚ࡣ࠸ࡃࡘሗ࿌ࡀ࠶ࡿࠋ ࡑࡢ㸯ࡘࠊAl ẘᛶᑐࡍࡿ Ca ࢜ࣥࡢᙺࡘ࠸࡚ࡢ◊✲ࡣྂࡃࡽ⾜ࢃࢀ
࡚࠸ࡿ (Z. Rengel 1992, Z. Rengel and W.-H. Zhang, 2003)ࠋAl ࡀ apoplasm
ධࡍࡿ⣽⬊ෆCa ࢜ࣥ⃰ᗘࡀୖ᪼ࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿࠋࡉࡽࠊAl ᛂ⟅
ࡉࢀࡿ࣮࢝ࣟࢫ (1,3- -glucan)ࡢㄏᑟࡸ㐣㓟ㄏᑟࠊ⣽⬊㦵᱁ࡢኚࡣ Ca ࡢᙳ㡪
㛵ಀࡀ࠶ࡿࡶゝࢃࢀ࡚࠸ࡿࠋ࣮࢝ࣟࢫࡣAl ࡼࡗ࡚ୖ᪼ࡋࡓ⣽⬊ෆ Ca ࡀᘬ
ࡁ㔠࡞ࡗ࡚1,3- -glucan synthase ࡀάᛶࡉࢀࠊ㞟✚ࡍࡿ (Krauss and Jeblick,
1991)ࠋࡋࡋࡇࡢ࣮࢝ࣟࢫࡣ Al ⤖ྜ⬟ࡀ࡞ࡃࠊࡢࡼ࠺࡞┠ⓗ࡛ྜᡂࡉࢀ࡚࠸ࡿ ࡣࡼࡃゎࡗ࡚࠸࡞࠸ࠋ㐣㓟ㄏᑟࡘ࠸࡚ࡣ᳜≀⣽⬊ࡢࡳ࡞ࡽࡎࠊື≀⣽⬊࡛ࡶ
⣽⬊ෆ Ca ࡀቑຍࡍࡿ㐣㓟ࢫࢺࣞࢫࡀ㉳ࡇࡿࡢሗ࿌ࡀ࠶ࡿ (Klyubin et al.
1996, Price et al. 1994)ࠋࡇࡢཎᅉ≀㉁ࡋ࡚ࡣ㉸㓟≀ (superoxide)ࡸ㐣㓟Ỉ
⣲ (hydrogen peroxide)ࡀ࠶ࡾࠊࢱࣥࣃࢡ㉁ࡢኚᛶࠊኚ␗ࠊගྜᡂ㜼ᐖࢆᘬࡁ㉳ࡇ ࡍࡉࢀ࡚࠸ࡿࠋࡋࡋࠊ⣽⬊ෆCa ࡀᘬࡁ㔠࠺ࡣ࡛᫂࠶ࡾࠊAl ࢫࢺࣞࢫ 㐣㓟ࢫࢺࣞࢫࡢ㛵㐃ᛶࡘ࠸࡚ࡶᮍゎ᫂ࡢ㒊ศࡀከ࠸ࠋࡲࡓࠊAl ࡼࡿᛂ ⟅ࡋ࡚⣽⬊㦵᱁ࡢኚࡀ࠶ࡿࠋࡇࢀࡽࡣᚤᑠ⟶ (microtubule)ࡸᚤᑠ⧄⥔ (microfilaments)ࡀᾘኻࡍࡿࡇࡼࡿࡶࡢ࡛ࠊࡇࡢ⤖ᯝࠊ⣽⬊ศࡸ⣽⬊ቨྜᡂ ࢆ㜼ᐖࡋ࡚࠸ࡿࡶࡢ⪃࠼ࡽࢀ࡚࠸ࡿࠋ࡞࠾ࠊࡇࡢᛂ⟅ࡣCa ࢩࢢࢼࣝࡢ࢝ࢫࢣ࣮ ࢻࡼࡿࡶࡢࡉࢀ࡚࠸ࡿࠋࡇࢀࡽCa ௨እࡢ Al ᛂ⟅ࡋ࡚ࠊ⣽⬊⭷ࡢ⾲㠃Ⲵ㟁 ࡢ⬺ศᴟࡸࠊ᰾ෆ࠾ࡅࡿAl-ࢡ࣐ࣟࢳࣥ⤖ྜయࡢ⏕ᡂࡸ⣳㗽యࡢᾘኻࡼࡿ⣽⬊ ศ㜼ᐖࠊࣜࢢࢽࣥࡢྜᡂ࡞ࡀ▱ࡽࢀ࡚࠸ࡿࠋ ᳜≀ࡣࡇࡢࡼ࠺࡞Al ࡼࡿ㞀ᐖࢆᅇ㑊ࡍࡿࡓࡵࠊ࠸ࡃࡘࡢ Al ⪏ᛶᶵᵓࢆᣢ
6
ࡗ࡚࠸ࡿ (Kochian et al. 2004)ࠋࡑࡢ 1 ࡘࡋ࡚ࠊ᰿➃ࡽ᭷ᶵ㓟ࢆᨺฟࡋ࡚ Al
ࢆ࣮࢟ࣞࢺࡋࠊ↓ẘࡍࡿᶵᵓࡀ࠶ࡿࠋᨺฟࡍࡿ᭷ᶵ㓟ࡣ᳜≀✀ࡼࡾ␗࡞ࡿࡀࠊ ࠼ࡤࢡ࢚ࣥ㓟ࢆᨺฟࡍࡿࡶࡢࡋ࡚ࢲࢬ (Pellet, D.M. et al. 1995)ࠊࢱࣂࢥ (Delhaize et al. 2001)ࠊࢽࣥࢪࣥ (Ojima et al. 1984)࡞ࡀ࠶ࡿࠋࡲࡓࠊࢥ࣒ࢠ (Delhaize et al. 1993)ࡸࢩࣟࢾࢼࢬࢼ (Hoekenga OA et al., 2003)ࡣࣜࣥࢦ㓟ࢆࠊ
ࢯࣂࡣࢩࣗ࢘㓟ࢆᨺฟࡍࡿࡇࡀ▱ࡽࢀ࡚࠸ࡿ (Zheng et al. 1998)ࠋ᭷ᶵ㓟ᨺฟ ࡢຠᯝࡣࡇࢀࡔࡅ࡛ࡣ࡞࠸ࠋ㓟ᛶ᮲௳ୗ࡛Al ⤖ྜࡋ࡚⁐ࡋࡓࣜࣥ㓟 Al ࡣࠊ ᭷ᶵ㓟ࡀᏑᅾࡍࡿAl ᭷ᶵ㓟࡛࣮࢟ࣞࢺࢆᙧᡂࡋࠊࣜࣥ㓟ࢆࡧ㐟㞳ࡉࡏࡿࡇ ࡀ࡛ࡁࡿࠋࡇࢀࡣࣜࣥ㓟Ḟஈࡢゎᾘࡶᙺ❧ࡘ (Ryan et al. 2001)ࠋࡇࡢࡼ࠺࡞ ሗ࿌ࡽࠊ᳜≀ࡼࡾከࡃࡢ᭷ᶵ㓟ࢆᨺฟࡉࡏࡿࡇࡀAl ⪏ᛶ࡞ࡿ⪃࠼ ࡽࢀࡿࡼ࠺࡞ࡗࡓࠋࡇࡢࡓࡵࡣࠊ᭷ᶵ㓟ྜᡂ㔞ࡢቑຍ᭷ᶵ㓟ᨺฟࢺࣛࣥࢫ࣏ ࣮ࢱ࣮ࡢⓎ⌧ቑຍࡢ2 ࡘࡀᚲせ࡞ࡿࠋ๓⪅ࡘ࠸࡚ࡣࠊ᭷ᶵ㓟ྜᡂ㓝⣲ࡢ㐣Ⓨ ⌧యࢆసᡂࡋࡓ࠸ࡃࡘࡢሗ࿌ࡀ࠶ࡿࠋFuente (1997)ࡽࡣࢡ࢚ࣥ㓟ྜᡂ㓝⣲ (Citrate Synthase, CS)ࢆࢱࣂࢥࡸࣃࣃ࡛ࣖ㐣Ⓨ⌧ࡉࡏࡓࡇࢁ Al ⪏ᛶ ࡞ࡗࡓሗ࿌ࡋ࡚࠸ࡿࠋࡲࡓࠊࣜࣥࢦ㓟ࢹࣄࢻࣟࢤࢼ࣮ࢮ (Malate dehydrogenase) ࢆ㐣Ⓨ⌧ࡋࡓࣝࣇࣝࣇ࡛ࡣࠊ᭷ᶵ㓟ྜᡂࡀቑຍࡋࡓࡔࡅ࡛ࡣ࡞ࡃࠊAl ࡶ⪏ᛶ࡞ࡗࡓ (Tesfaye et al. 2001)ࠋ࠶ࡿ࠸ࡣ CS ࢆࢩࣟࢾࢼࢬࢼ࡛㐣Ⓨ⌧ ࡉࡏࡓࡇࢁࠊࣜࣥ㓟Al ࡼࡿపࣜࣥ㓟᮲௳࡛ࡢ⏕⫱ࡀᨵၿࡋࡓ࠸࠺ሗ࿌ࡶ࠶ ࡿ (Koyama et al. 2000)ࠋ୍᪉ࠊᚋ⪅ࡘ࠸࡚᭷ᶵ㓟ᨺฟࡢࢽ࢜ࣥࢳࣕࢿࣝࡢ᥈ ⣴ࡀ⾜ࢃࢀ࡚ࡁࡓࠋࣃࢵࢳࢡࣛࣥࣉἲ࡞ࡼࡾࠊAl ᛂ⟅ࢽ࢜ࣥࢳࣕࢿࣝࡢ◊ ✲ࡀ⾜ࢃࢀ࡚ࡁࡓ (Kochian et al. 2001)ࡀࠊࡑࡢാࡁࢆࡍࡿ㑇ఏᏊࡣࡇࢀࡲ࡛༢㞳 ࡉࢀ࡚ࡇ࡞ࡗࡓࠋࡋࡋ㏆ᖺࠊࢥ࣒ࢠ࠾࠸࡚Al ࡼࡗ࡚άᛶࡉࢀࡿࣜࣥࢦ
㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࠊALMT1 (Aluminum activated malate transporter)ࡀ༢㞳ࡉ
7
ࢀࡓ (Delhaize et al., 2004)ࠋࡲࡓࠊࢩࣟࢾࢼࢬࢼ࡛ࡶࣜࣥࢦ㓟ࢺࣛࣥࢫ࣏࣮ࢱ
࣮ࡀぢࡘࡾ (Hoekenga OA et al., 2006)ࠊࡉࡽࢡ࢚ࣥ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡀࢶ
ࣝࢬ࢟ (Vigna umbellate)ࡽⓎぢࡉࢀࡓ (Jian Li Yang et al., 2006)ࠋᚋࡣ
᭷ᶵ㓟ྜᡂࠊᨺฟࡢ㠃ࡽAl ⪏ᛶࡢࡀᮇᚅࡉࢀࡿࠋ
ࡲࡓࠊpH ࡍ࡞ࢃࡕ H+ࡢᐖࡶྠᏑᅾࡍࡿࠋከࡃࡢရ✀ࡀᏑᅾࡍࡿࢩࣟࢾ
ࢼࢬࢼ࠾࠸࡚ Al ప pH ࡛ప pH ࡢࡳឤཷᛶࢆ♧ࡍရ✀ࡀሗ࿌ࡉࢀ࡚࠸ࡿ
(Ikka et al., 2007)ࠋࡇࢀࡣ Al pH ࡢᐖࡣ␗࡞ࡿᶵᵓ࡛࠶ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿ
ࡀࠊࡇࡢᕪࡘ࠸࡚ࡢヲࡋ࠸ሗ࿌ࡣࡇࢀࡲ࡛࡞࠸ࠋࡇࢁ࡛ࠊపpH ឤཷᛶࢆ
♧ࡍࢩࣟࢾࢼࢬࢼࡋ࡚㑅ᢤࡉࢀ࡚ࡁࡓኚ␗యࡣࡑࡢᚋࠊzinc finger protein ㌿
ᅉᏊࡢኚ␗࡛࠶ࡿࡇࡀ♧ࡉࢀࠊAtSTOP1 (sensitive to proton rhizotoxicity 1)
ྡࡅࡽࢀࡓࠋࡇࡢኚ␗యࡣ Al ࡶឤཷᛶ࡛࠶ࡗࡓࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ (Iuchi et al., 2007)ࠋࡋࡋࠊࡇࡢ㌿ᅉᏊࡀไᚚࡋ࡚࠸ࡿୗὶࡢ㑇ఏᏊࡘ࠸࡚ ࡣㄪࡽࢀ࡚࠸࡞࠸ࠋ ᮏ◊✲࡛ࡣࠊSTOP1 ㌿ᅉᏊࢆ୰ᚰࡋࡓ Alࠊప pH ⪏ᛶᶵᵓࡢゎ᫂ࢆ┠ⓗ ࡋࡓࠋ➨1 ❶࡛ࡣࡇࡢ㌿ᅉᏊࡼࡗ࡚ไᚚࡉࢀ࡚࠸ࡿୗὶ㑇ఏᏊࡢ᥈⣴ࢆ⾜ࡗ ࡓࠋSTOP1 ࡀᶵ⬟ࡋ࡞࠸ࡇ࡛ Al ࡸప pH ឤཷᛶ࡞ࡿ࠸࠺ࡇࡣࠊSTOP1 ࡀไᚚࡋ࡚࠸ࡿ㑇ఏᏊࡀാ࡞ࡃ࡞ࡗࡓ (ࡶࡋࡃࡣാ࠸࡚ࡋࡲࡗࡓ)⤖ᯝࠊឤཷᛶ ࡞ࡗࡓࠊ࠸࠺ࡇ࡛࠶ࡿࠋAl ࡸప pH ࡢ⪏ᛶᶵᵓࡣ」ྜⓗ࡞ࡶࡢ࡛࠶ࡾࠊࡑ ࡢࡼ࠺࡞㑇ఏᏊࢆ᥈⣴ࡍࡿࡓࡵࡣࠊࢤࣀ࣒⛉Ꮫⓗᡭἲࡼࡾ㑇ఏᏊࡍ࡚ࢆ⥙⨶ ⓗㄪࡿࡇࡀᚲせ࡞ࡿࠋࡇࢀࢆ⾜࠺ࡓࡵࡢᙉຊ࡞ࢶ࣮࣐ࣝࢡࣟࣞࡀ ࠶ࡿࠋ࣐ࢡࣟࣞࡣከࡃࡢ㑇ఏᏊࡢ㌿ゎᯒࢆ⥙⨶ⓗ⾜࠺ࡇࡀ࡛ࡁࡿࡓࡵࠊ ࢤࣀ࣒యࡢゎᯒ㠀ᖖ᭷⏝࡛࠶ࡾࠊࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ゎᯒ࠸ࢃࢀࡿࠋ㏆ ᖺࡣ⳦㢮ࠊື≀ࠊ᳜≀ᵝࠎ࡞⏕≀✀ࡢࢤࣀ࣒㓄ิࡀゎㄞࡉࢀ࡚ࡁࡓࡇࡽᗈࡃ ࢃࢀࡿᡭἲ࡞ࡾࠊ㑇ఏᏊ᥈⣴ࡢ㔜せ࡞ࢶ࣮ࣝࡋ࡚⏝ࡉࢀ࡚࠸ࡿࠋ≬≟࢘
8 ࣝࢫᑐࡍࡿᢠ࢘ࣝࢫ⸆ࡢᛂ⟅㑇ఏᏊࡢ᥈⣴ࡶ⏝ࡉࢀ࡚࠸ࡿ (Masatani et al., 2010)ࠋ᳜≀࠾࠸࡚ࠊࣔࢹ᳜ࣝ≀ࡢࢩࣟࢾࢼࢬࢼࡢ࣐ࢡ࡛ࣟࣞࡣࠊ ࡰࡍ࡚ࡢ㑇ఏᏊࡀྵࡲࢀ࡚࠸ࡿࠋAl ࢫࢺࣞࢫࡢࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ゎᯒ ࡼࡾࠊࣜࣥࢦ㓟ྜᡂࠊ࠾ࡼࡧࡑࡢ࿘㎶ࡢ௦ㅰ㑇ఏᏊࡢኚືࢆ᫂ࡽࡋࡓ (⃝ᮌ et al., 2008)ࠋࡲࡓࠊAlࠊሷࠊCdࠊCu ࡢࢫࢺࣞࢫࢆ࣐ࢡࣟࣞࡽ≉␗ⓗᛂ ⟅ࡍࡿ㑇ఏᏊࢆ᫂ࡽࡋ (Zhao, et al., 2009)ࠊࡇࢀࡽࢫࢺࣞࢫࡢⅣ⣲௦ㅰࡸ࣑
ࣀ㓟௦ㅰ⤒㊰ࡢࣉࣟࣇࣝࢆゎᯒࡋࡓ (Zhao, et al., 2010)ࠋࡇࡢࡼ࠺ࠊSTOP1
㌿ᅉᏊࡸAl ࡸ pH ࡘ࠸࡚ࡢࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ゎᯒࡽ STOP1 ୗὶ㑇ఏ
Ꮚࢆ᥈⣴ࡋ࡚࠸ࡃࠋ➨2 ❶࡛ࡣࠊ⏘ᴗ᳜≀࡛࠶ࡿ࣮ࣘ࢝ࣜࢆᮦᩱࠊAlࠊప pH ⪏
ᛶᶵᵓࡀࣔࢹ᳜ࣝ≀࡛࠶ࡿࢩࣟࢾࢼࢬࢼඹ㏻ࡢࡶࡢ࡞ࡢࠊࡲࡓࢩࣟࢾࢼࢬ
ࢼSTOP1 ࡢ࣍ࣔࣟࢢࡀᏑᅾࡋࠊࡘࡑࡢୗὶ Al ⪏ᛶ㑇ఏᏊࡀᏑᅾࡍࡿࡢㄪ
9
➨
➨
1 ❶
ࢩࣟࢾࢼࢬࢼࡢ
Alࠊప pH ⪏ᛶ㌿ᅉᏊ STOP1
ࡀไᚚࡍࡿୗὶ㑇ఏᏊࡢ᥈⣴
1-1 ⥴ゝ
᳜≀ࡣࠊ⇱ࠊሷࠊ㓟ᛶᅵተ࡞ࠊᵝࠎ࡞⎔ቃࢫࢺࣞࢫ㐺ᛂࡍࡿࡓࡵ⪏ᛶ࣓࢝ࢽࢬ࣒ࢆാࡏ࡚࠸ࡿ (Seki et al., 2003; Kochian et al., 2004)ࠋࡇࡢࡼ࠺࡞
ࢩࢫࢸ࣒ࢆศᏊ⏕≀ᏛࡢほⅬ࡛ゎ᫂ࡍࡿࡇࡼࡾࠊࢫࢺࣞࢫ⎔ቃୗ࡛࠶ࡗ࡚ࡶ㧗 ⏕⏘࡛ࡁࡿస≀ࢆసฟࡍࡿࡓࡵࡢရ✀ᨵⰋࢆຠ⋡ⓗⓎᒎࡉࡏࡿࡇࡀᮇᚅ࡛ࡁ
ࡿࠋࡇࡢࡼ࠺࡞⫼ᬒ࠾࠸࡚ࠊ㓟ᛶᅵተ࡛㉳ࡇࡿ᳜≀᰿ࡢAl3+ࠊH+ࡢẘᛶ (Kinraide,
2003)ࠊ࣐ࣥ࢞ࣥ㐣 (Horst, 1988)ࠊࣜࣥ㓟Ḟஈ (Neumann et al., 1999)ࠊ࡞ ࡢࢫࢺࣞࢫᑐࡍࡿ⪏ᛶ࣓࢝ࢽࢬ࣒ࡣࠊရ✀ᨵⰋ࠾ࡅࡿ㔜せ࡞ࢱ࣮ࢤࢵࢺ࡞ࡿࠋ ࡑࢀࡣࠊ㓟ᛶᅵተࡀୡ⏺ࡢ⪔సྍ⬟࡞ᅵᆅࡢ 40㸣ࢆ༨ࡵ࡚࠾ࡾࠊ⏕⫱㜼ᐖࢆ㉳ࡇ ࡋ࡚࠸ࡿࡓࡵ࡛࠶ࡿࠋ Al ⪏ᛶᛂ⟅ࡍࡿ࠸ࡃࡘࡢ⪏ᛶ㑇ఏᏊࡣࠊᵝࠎ࡞✀ࡢ᳜≀ࡽ༢㞳ࡉࢀ࡚࠸ ࡿࠋAl3+ࢆ㝖ࡍࡿ࣓࢝ࢽࢬ࣒ࡢ㔜せ࡞ᶵᵓࡋ࡚᭷ᶵ㓟ᨺฟࡀ࠶ࡾࠊࢡ࢚ࣥ㓟ࡸ ࣜࣥࢦ㓟ࢆᨺฟࡍࡿࠋࡑࡢᶵ⬟ࢆᣢࡘ㑇ఏᏊࡋ࡚᭷ᶵ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡀ࠶ࡾࠊ
ࣜࣥࢦ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡣࢥ࣒ࢠ (Triticum aestivum; TaALMT1 [Sasaki et
al., 2004])ࡸࢩࣟࢾࢼࢬࢼ (Arabidopsis thaliana; AtALMT1 [Hoekenga et al., 2006])ࡽࠊࢡ࢚ࣥ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡣࣔࣟࢥࢩ (Sorghum bicolor; Magalhaes
et al., 2007)ࡽྠᐃࡉࢀࡓࠋࠊኚ␗య◊✲ࡽ༢㞳ࡉࢀࡓ ALS3(Aluminum
Sensitive 3)ࡣࠊhalf type ATP-binding cassette transporter ࢆࢥ࣮ࢻࡋࠊAl ឤཷ
ᛶࡢ⤌⧊ࡽ Al ࢆ㓄ศࡍࡿ⤒㊰ࡢ㛵ࢃࡾࡀ♧၀ࡉࢀ࡚࠸ࡿ (Larsen et al.,
10
ຠ⋡ⓗ࡞ࣜࣥ㓟⋓ᚓࢆไᚚࡍࡿ㘽࡞ࡿ㑇ఏᏊࡀྠᐃࡉࢀࡓ (Mitsukawa et al.,
1997)ࠋࡋࡋࠊH+ࡣᵝࠎ࡞᳜≀ᑐࡋ࡚㧗࠸ẘᛶࢆ♧ࡍࡶࢃࡽࡎ (Koyama
et al., 1995; Yokota and Ojima, 1995; Kinraide, 2003; Watanabe and Okada, 2005)ࠊ࣐ࣥ࢞ࣥẘᛶ࡞ࡢ㓟ᛶᅵተࡢࢫࢺࣞࢫẚ㍑ࡋ࡚ࠊࡑࡢศᏊ⏕≀Ꮫⓗ ▱ぢࡣࢃࡎࡋ࡞࠸ (Delhaize et al., 2003)ࠋ ᰿ẘᛶࡢᐇ㦂࠾࠸࡚ࠊH+Al ࡛യᐖࣃࢱ࣮ࣥࡣ␗࡞ࡗ࡚࠾ࡾ (Koyama et al., 1995)ࠊࡉࡽࠊ㓟ᛶ᳃ᯘᅵተࡢ᰿ఙ㛗ࡢࣔࢹࣜࣥࢢࡼࡗ࡚ࠊࡇࢀࡽ 2 ࡘࡢ ࢫࢺࣞࢫせᅉࡣ༊ูࡉࢀࡓ (Kinraide, 2003)ࠋࡇࢀࡽࡢሗ࿌ࡣࠊH+Al ࡢ᰿ẘᛶ ࡀࠊ␗࡞ࡿࣉࣟࢭࢫࡼࡗ࡚യᐖࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋࡋࡋࠊ Al ⪏ᛶရ✀ࢆ⏝࠸ࡓ࠸ࡃࡘࡢᐇ㦂࡛ࡣࠊᩘ✀ࡢ᳜≀✀࠾࠸࡚ࠊH+⪏ᛶ Al
⪏ᛶࡣ㑇ఏᏛⓗ㛵㐃ࡋ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡓ (Rangel et al., 2005; Yang et
al., 2005)ࠋࡇࢀࡽࡢሗ࿌ࡣࠊH+Al ࡢẘᛶ࣓࢝ࢽࢬ࣒ࡀ␗࡞ࡗࡓ⏕≀Ꮫⓗࣉࣟࢭ ࢫ࡞ࡗ࡚࠸ࡿࡀࠊ࠸ࡃࡘࡢ᳜≀✀࡛ࡣࠊH+Al ࡢࢫࢺࣞࢫ⪏ᛶࡀሙྜࡼࡗ ࡚ࡣྠࡌ㑇ఏᅉᏊ࡛ྠㄪ⠇ࡉࢀ࡚࠸ࡿྍ⬟ᛶࢆ♧ࡋ࡚࠸ࡿࠋ㏆ᖺࠊෆࡽࡣ H+ẘᛶAl ẘᛶ㉸ឤཷᛶࢆ♧ࡍࢩࣟࢾࢼࢬࢼstop1 ኚ␗యࢆ༢㞳ࡋࡓ (Iuchi et al., 2007)ࠋࡇࡢኚ␗యࡣศᏊ࡛ࣞ࣋ࣝࡢ H+⪏ᛶ࣓࢝ࢽࢬ࣒ࡸࠊH+Al ࡢ⪏ᛶ ࣓࢝ࢽࢬ࣒ࡢ┦㛵ಀࢆゎᯒࡍࡿࡓࡵࡢࡼ࠸ࣔࢹࣝ࡞ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡇࡢࢩࣟ ࢾࢼࢬࢼstop1 ኚ␗యࡣࠊ࢚ࢳ࣓ࣝࢱࣥࢫࣝࣇ࢛ࣥ㓟ࡼࡗ࡚✺↛ኚ␗ࢆㄏⓎࡋ
ࡓ㞟ᅋࡽࠊపpH ᐮኳᇵᆅࢆ㑅ᢤᣦᶆࡋࡓ root bending assay ࡢ⤖ᯝࠊ᰿ఙ㛗
ࡀ㜼ᐖࡉࢀࡓಶయࡋ࡚༢㞳ࡉࢀࡓ (Iuchi et al., 2007)ࠋ⯆῝࠸ࡇࠊstop1
ኚ␗యࡣH+ឤཷᛶࡋ࡚༢㞳ࡉࢀࡓࡶࢃࡽࡎࠊAl ᑐࡋ࡚ࡶ㉸ឤཷᛶࢆ♧
ࡋࡓࠋࡑࡢᚋࡢࢤࣀ࣒DNA 㓄ิゎᯒ࡛࠶ࡿ Chromosome-walking ࡢゎᯒࡼࡾࠊ
stop1 ኚ␗యࡣ zinc finger protein ࡢࡶࡗࡶ㔜せ࡞ Cys-2-His-2 ࣔࢳ࣮ࣇࡢ His
11
Al ⪏ᛶࡢ㔜せ࡞㑇ఏᏊࡢ 1 ࡘ࡛࠶ࡿࣜࣥࢦ㓟ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࠊAtALMT1
(Hoekenga et al., 2006)ࡶstop1 ኚ␗య࡛㌿㔞ࡀᢚไࡉࢀ࡚࠸ࡓ (Iuchi et al.,
2007)ࠋࡇࡢ stop1 ኚ␗యࢩࣟࢾࢼࢬࢼ࡛ࡣࠊ㔜せ࡞ Al ⪏ᛶ࣓࢝ࢽࢬ࣒࡛࠶ࡿ
Al ᛂ⟅ࣜࣥࢦ㓟ᨺฟࡀṆࡋ࡚࠸ࡿ (Kobayashi et al., 2007)ࠋࡋࡋࠊࣀࢵࢡ
࢘ࢺኚ␗య (T-DNA ᤄධࣛࣥ)ࢆ⏝ࡋࡓ㏫㑇ఏᏛࡢᐇ㦂࡛ࡣࠊH+ࡣឤཷᛶࢆ
♧ ࡉ ࡎ ࠊAtALMT1 ࡀ H+⪏ ᛶ ࣓ ࢝ ࢽ ࢬ ࣒ ࡣ ྵ ࡲ ࢀ ࡞ ࠸ ࡇ ࡀ ♧ ࡉ ࢀ ࡓ
(Kobayashi et al., 2007)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊSTOP1 ࡀ H+Al ࡢ㉸ឤཷᛶỴᐃ
ⓗᙳ㡪ࢆ࠼ࡿࡢ㑇ఏᏊࡢⓎ⌧ࢆㄪ⠇ࡍࡿᶵ⬟ࢆᣢࡘࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ㏆
ᖺࠊLiu et al. (2009)ࡽࡣࠊࢩࣟࢾࢼࢬࢼࡢ Al ⪏ᛶ㛵ࢃࡿࠊmultidrug and toxic
compound exclusion protein ([AtMATE]; At1g51340)ࡢࡼ࠺࡞㑇ఏᏊࡶ STOP1
ࡼࡗ࡚ไᚚࡉࢀ࡚࠸ࡿሗ࿌ࡋࡓࠋzinc finger protein ࡣᵝࠎ࡞ᶵ⬟ࢆᣢࡗ࡚࠸ࡿ
ࡀ (Englbrecht et al., 2004)ࠊࡇࢀࡽࡢ⤖ᯝࡣ STOP1 ࡀ Al ࡸ H+ࡢ⪏ᛶ࣓࢝ࢽࢬ
࣒ࢆไᚚࡍࡿࠊሗఏ㐩⤒㊰῝ࡃࢃࡿ㔜せ࡞ᅉᏊ࡛࠶ࡿࡇࢆ♧ࡋࠊAl ⪏
ᛶࡢAtALMT1 (Iuchi et al., 2007)ࡸࠊAtMATE (Liu et al., 2009)௨እ࡛ࡶᵝࠎ࡞ ⪏ᛶ㑇ఏᏊࢆไᚚࡋ࡚࠸ࡿྍ⬟ᛶࡀ࠶ࡿࠋ
ࡇࡢ◊✲࡛ࡣࠊstop1 ኚ␗య࠾ࡅࡿ Al H+ࡢ㉸ឤཷᛶࡢศᏊ⏕⌮Ꮫⓗ࡞≉
ᚩ࡙ࡅࢆ⾜ࡗࡓࠋDNA ࣐ࢡࣟࣞゎᯒࡣࠊAtALMT1 ࡸ ALS3 ࡢࡼ࠺࡞ Al
⪏ᛶ㔜せ࡞㑇ఏᏊࡸࠊపpH ₯ᅾⓗ㈉⊩ࡍࡿࠊ࣒࢝ࣜ࢘ࡢࡼ࠺࡞࢜ࣥᜏᖖ
ᛶࡢ⏕⌮㐣⛬ᒓࡍࡿ㑇ఏᏊࡀࠊstop1 ኚ␗య࡛」ᩘࠊ㌿ᢚไࡉࢀࡿࡇࢆ᫂ࡽ
ࡋࡓࠋຍ࠼࡚ࠊ௦ㅰ≀ࡸ㌿≀ࡢ⥲ྜⓗ࡞ゎᯒ࡛ࡣࠊ᳜≀ࡢ⣽⬊㉁ෆpH ไᚚ
ࡉࢀࡿ௦ㅰ⤒㊰ࠊbiochemical pH stat (Sakano, 1998)ࡸ -aminobutyric acid [GABA] shunt (Bouche and Fromm, 2004)⤒㊰ࡢ㑇ఏᏊࠊ௦ㅰ⏘≀ࡶኚ␗య࡛ᢚ
ไࡉࢀ࡚࠸ࡿࡇࢆ᫂ࡽࡋࡓࠋ⯆῝࠸ࡇࠊ࢜ࣥᜏᖖᛶpH ไᚚ௦ㅰ
12 pH ⪏ᛶ㔜せ࡛࠶ࡿࡇࡽࠊ㧗➼᳜≀࡛ࡶࡑࡢྍ⬟ᛶࡣ࠶ࡿ (Yan et al., 1992)ࠋ ࡇࢀࡽࡢ⤖ᯝࡣࠊSTOP1 ࡀ㓟ᛶᅵተࡢ࡞ࢫࢺࣞࢫせᅉࡢ」ྜⓗ࡞⪏ᛶ࣓࢝ࢽࢬ ࣒ࢆไᚚࡋࠊstop1 ኚ␗య࡛㌿ᢚไࡉࢀࡓ H+⪏ᛶࢩࢫࢸ࣒ࡢᩘ✀㢮ࡀࡢ⏕≀ ✀㢮ఝࡋ࡚࠸ࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋ
11-2 ᐇ㦂ᮦᩱཬࡧᐇ㦂᪉ἲ
ࢩࣟࢾࢼࢬࢼࢡࢭࢵࢩࣙࣥࢩࣟࢾࢼࢬࢼstop1 ኚ␗యࡣࠊ࢚ࢥࢱࣉ Col-0 ࢆ ethyl methanesulfonate
ฎ⌮࡛ኚ␗ㄏᑟࡉࡏࡿࡇࡼࡗ࡚ᚓࡽࢀࡓF2 㞟ᅋ࡛࠶ࡿ (Iuchi et al., 2007)ࠋ
AtALMT1-KO (SALK_009629) ࠊ STOP1-KO (SALK_114108) ࠊ ALS3-KO (SALK_061074) ࡢ SALK T-DNA ᤄ ධ ࣀ ࢵ ࢡ ࢘ ࢺ ኚ ␗ య ࡣ ࠊ Arabidopsis Biological Resource Center ࡽධᡭࡋࡓࡶࡢ࡛࠶ࡿࠋCol-0 (JA58) and Col-4 (N933)ࡣࠊࡑࢀࡒࢀ RIKEN Bio-Resource Center Nottingham Arabidopsis Stock Center ࡽධᡭࡋࡓࡶࡢ࡛࠶ࡿࠋSTOP1 ┦⿵⤌యࡣࠊstop1 ኚ␗య CaMV35S ࣉ࣮ࣟࣔࢱ࣮ࢆ᥋⥆ࡋࡓ STOP1 ࣋ࢡࢱ࣮ (CaMV35S::STOP1)ࢆᑟධ ࡋࡓ⤌య࡛࠶ࡾࠊෆࡽࡼࡗ࡚సᡂࡉࢀࡓࡶࡢ࡛࠶ࡿ (Iuchi et al., 2007)ࠋ Ỉ⪔᱂ᇵ࠾ࡼࡧ⏕⫱ヨ㦂 Ỉ⪔⏕⫱ヨ㦂ࡣࠊෆࡽࡢሗ࿌ྠ᮲௳࡛⾜ࡗࡓ (Iuchi et al., 2007)ࠋᗂ᳜≀ యࡣᨵኚMGRL ᇵᆅ (1/50 ᙉᗘࠊPi 㝖ཤࠊ200 M CaCl2ࠊpH 5.5)ࢆᇶᮏࠊྛ ࢫࢺࣞࢫࡢ⁐ᾮࢆฎ⌮⃰ᗘ࡞ࡿࡼ࠺ῧຍࡋࡓࠋ⁐ᾮࡣ1 ᪥࠾ࡁ᭦᪂ࡋࠊ᰿㛗 ࢆ ᐃࡋࡓࠋ⏕⫱ࡣ12 㛫᫂ᮇࠊගᗘ 250 mol m-2 s-1ࠊ25 Υ࡛ 7 ᪥㛫⏕⫱ࡋࡓࠋ ᰿㛗ヨ㦂ࡣ㔝⏕ᆺࣀࢵࢡ࢘ࢺኚ␗యࡢᐇ⏕ࡑࢀࡒࢀ10 ಶయࡢ࠺ࡕࠊୖ 3 ಶ
13 యࡢ᰿㛗ࢹ࣮ࢱࢆ⏝ࡋࡓࠋ୍᪉ࠊCaMV35S::AtALMT ࢆstop1 ኚ␗యᑟධࡋ ࡓ┦⿵⤌యࡣ20 ಶయࢆ⏝ࡋࡓࠋ ᅵ ᅵ⪔ヨ㦂ἲ ᅵ⪔⏝ࡋࡓᅵࡣࠊᮾᏛᏛ㝔㎰Ꮫ◊✲⛉㝃ᒓ」ྜ⏕ែࣇ࣮ࣝࢻᩍ⫱ ◊✲ࢭࣥࢱ࣮ (ᕝΏࢭࣥࢱ࣮) ࡛᥇ྲྀࡉࢀࡓ㓟ᛶ㯮࣎ࢡᅵࢆ⏝ࡋࡓࠋࡇࡢ㯮࣎ࢡ ᅵࡣࠊ᭷ᶵ≀ࡶẘᛶ࣑ࣝࢽ࣒࢘ࡀከࡃྵࡲࢀ࡚࠸ࡿࠋᑐ↷࡞ࡿ〓Ⰽ᳃ᯘ ᅵ (୰ᛶࠊ↓ẘᛶ࣑ࣝࢽ࣒࢘) ࡣⶶ⋤࡛ྲྀᚓࡋࡓࠋࡇࢀࡽࡢᅵተࡢ≉ᛶࡣㄪࡽ
ࢀ࡚࠾ࡾࠊAlࠊࣉࣟࢺࣥࡢ᳜≀ࡢᐖࡶㄪࡽࢀ࡚࠸ࡿ (Ikka et al., 2007)ࠋᅵ⪔
ヨ㦂ࡣࠊࡑࢀࡒࢀࡢᅵተከ㔞ඖ⣲ࠊᚤ㔞ඖ⣲ࢆῧຍࡋ (Kobayashi et al., 2005)ࠊ
ࡉࡽ㯮࣎ࢡᅵࡣCaCO3 (60, 250, and 400 mg per 100 g of dry soil) ࡛ẁ㝵ⓗ
୰ࡋࡓࠋᅵተpH (Shoji et al., 1964) ࠾ࡼࡧ ᛶ Al ࢆ ᐃࡋࡓ (Koyama
et al., 2000)ࠋ
GFP ࡢ୍㐣ⓗⓎ⌧ゎᯒ
GFP (sGFP) ࡣࠊ᳜≀ࡢࢩࢫࢸ࣒㐺ྜࡋࡓࢥࢻࣥ࡞ࡿࡼ࠺㓄ิࢆ᭱㐺 ࡋࡓࡶࡢࢆ⏝ࡋࡓ (Chiu et al., 1996)ࠋࡇࡢ GFP ࢆ AtSTOP1 㓄ิࡢ N ᮎ➃
᥋⥆ࡍࡿࡓࡵࠊoverlap PCR ࡛ GFP AtSTOP1 ࢆ᥋⥆ࡋࡘࡘቑᖜࡋࡓࠋGFP ࡣ
pBE2113 (containing sGFP) ࢆ 㗪 ᆺ ࠊ SfiI-GFP
(5’-CATTTGGCCAAATCGGCCATGGTGAGCAAGGGCGAGGAGCTG-3’), GFP-NotI
(5’-GGATTAGCGGCCGCCCTTGTACAGCTCGTCCATGCCGTGAG-3’) ࡢࣉ࣐࣮ࣛ
࡛ቑᖜࡋࡓࠋࡲࡓࠊAtSTOP1 ࡣࠊRAFL (the RIKEN full-length cDNA clone
[RAFL09-20I22; Seki et al., 2002] ࢆ 㗪 ᆺ ࠊ NotI-STOP1
14
(5’-ACAAGGGCGGCCGCATGGAAACTGAAGACGATTTGTGCAAC-3’) ࡢࣉ࣐࣮ࣛ
࡛ቑᖜࡋࡓࠋࡇࡢቑᖜࡋࡓDNA ᩿∦ࢆࡑࢀࡒࢀไ㝈㓝⣲ SfiI NotI ࡛ษ᩿ࡋࠊ
ྠᵝSfiI ࡛ษ᩿ࡋࡓ pBI2113SF ࣋ࢡࢱ࣮ᑟධࡋࡓࠋ㓄ิࡣ DNA ࢩ࣮ࢣࣥࢧ
࣮࡛☜ㄆࡋࡓࠋࡇࡢ࣋ࢡࢱ࣮ࢆࢱ࣐ࢿࢠࡢ⾲⓶⣽⬊ࣃ࣮ࢸࢡࣝ࢞ࣥἲ࡛ᑟධࡋࠊ
⣽⬊ࢆ22Υ࡛ 12 㛫࣮ࣥ࢟ࣗ࣋ࢺᚋࠊGFP ࡢ⺯ගࢆほᐹࡋࡓ (Fujita et al.,
2004)ࠋ
sstop1 ኚ␗యࡢ AtALMT1 ┦⿵ヨ㦂
AtALMT1 ࡢ DNA ᩿∦ࡣࠊࢩࣟࢾࢼࢬࢼࡢࢤࣀ࣒ DNA ࢆ㗪ᆺ PCR ࡛ቑ
ᖜ ࡋ ࡓ ࠋ ቑ ᖜ ⏝ ࡋ ࡓ ࣉ ࣛ ࣐ ࣮ ࡣ ࠊ Forward:
5’-CATTTGGCCAAATCGGCCATGGAGAAAGTGAGAGAGATAGTGAGAGAAG-3’ ࠊ
Reverse: 5’-AGAACGGCCTTATGGCCTTACTGAAGATGCCCATTACTTAATG-3’࡛࠶
ࡿࠋAtALMT1 ࡣࠊࣂࢼ࣮ࣜ࣋ࢡࢱ࣮pBE2113SF (Liu et al., 1998)ࡢ CaMV 35S
ࣉ࣮ࣟࣔࢱ࣮ࡢୗὶᤄධࡋࡓࠋࡇࡢ࣋ࢡࢱ࣮ࢆࠊstop1 ኚ␗యࡢࢩࣟࢾࢼࢬࢼ
floral dip ἲ (Clough and Bent, 1998) ࡛ᑟධࡋࠊᚓࡽࢀࡓ⤌యࡣࠊT1 ᳜≀
యࢆ⮬ᐙཷ⢭ࡉࡏࠊT2 ୡ௦ࢆᚓࡓࠋ
RNA ࠾ࡼࡧ࣓ࢱ࣮࣒࣎ࣟゎᯒࢧࣥࣉࣝㄪ〇
ᐇ⏕ࢆ200 M CaCl2 ධࡾ 1/50 ᙉᗘ MGRL Ỉ⪔ᾮ (pH 5.6) ✀ࡋ (Toda
et al., 1999)ࠊ30 mol quanta m-2 s-1, 12 㛫᫂ᮇ, 25Υࡢ⎔ቃ࡛ࠊ⁐ᾮࡣ 1 ᪥࠾
ࡁ᭦᪂ࡋࡓࠋ10 ᪥┠ࡢᗂ᳜≀యࢆ Al ⁐ᾮ (10 M AlCl3 pH 5.0)ࠊప pH ⁐ᾮ
(pH4.5) ⛣ࡋࡓ (Iuchi et al., 2007)ࠋฎ⌮୰ࡣ㐃⥆↷᫂ࡋࠊ⛣᳜ 24 㛫ᚋ
᰿ࢆࡣࡉࡳ࡛ษ᩿ࡋࠊᾮయ❅⣲࡛⣲᪩ࡃ⤖ࡋࠊ-80Υ࡛ಖᏑࡋࡓࠋRNA ࡣ㕥ᮌࡽ
15
Agilent 2100 Bioanalyzer ࢆ ⏝ ࡋ ࡚ ศ ゎ ⛬ ᗘ ࢆ ุ ᐃ ࡋ ࡓ ࠋ GC-MS (gas chromatography-mass spectrometry) CE-MS (capillary electrophoresis-mass spectrometry) ࡢ௦ㅰ≀ ᐃ⏝ࡍࡿࢧࣥࣉࣝࡣࠊ50% ࢚ࢱࣀ࣮ࣝ Qiagen Mixer Mill MM300 kit ࡛ᢳฟࡋࡓࠋࡑࡢᚋࠊGC-TOF-MS (for GC-time of flighty-mass spectrometry)ࠊCE-MS ࡢ㝧࢜ࣥᛶࠊ㝜࢜ࣥᛶศ㞳⏬ศࢆ N-methyl-N-(trimethylsilyl)-trifluoroacetamide ࡛ ๓ ฎ ⌮ ࡋ ࡓ (Urano et al., 2009)ࠋ R RT-PCR ࠾ࡼࡧᐃ㔞 RT-PCR RT-PCR ࡣࠊᑠᯘࡽࡢ᪉ἲ࡛⾜࠸ (Kobayashi et al., 2007)ࠊᐃ㔞 RT-PCR ࡣ ࣜࣝࢱ࣒PCR (Applied Biosystems 7300) ࢆࣉࣟࢺࢥࣝᚑ࠸⏝ࡋࡓࠋ㑇 ఏᏊࡢ㌿㔞ࡣࠊࣘࣅ࢟ࢳࣥ UBQ1 ࡢ㌿㔞ࢆࢥࣥࢺ࣮ࣟࣝࡋ࡚ᶆ‽ࡋࡓࡢ ࡕࠊ㔝⏕ᆺẚ㍑ࡋࡓࠋࣉ࣐࣮ࣛ㓄ิࡣࠊTable 1 ♧ࡋࡓࠋ ࣐ࢡࣟࣞ࠾ࡼࡧࢹ࣮ࢱゎᯒ ࣐ࢡࣟࣞࡣAgilent ࡢ࣐ࢡࣟࣞࢳࢵࣉ Arabidopsis 3 ࢆࣉࣟࢺࢥ ࣝᚑ࠸ࠊCy3 Cy5 ࡢ 2 Ⰽἲ࡛⾜ࡗࡓࠋࣁࣈࣜࢲࢮ࣮ࢩࣙࣥࡢ⤌ྜࡏࡣࠊ Col-4 ࡢࢥࣥࢺ࣮ࣟࣝ (-Al, pH 5.0, 24 h) ࢫࢺࣞࢫฎ⌮ (-Al, pH 4.5, 24 h ࡲࡓ ࡣ 10 M Al solution, pH 5.0, 24 h)ࠊAlࠊప pH ࢫࢺࣞࢫฎ⌮ࡢ Col-0 stop1
ኚ␗యࠊ࠾ࡼࡧAl ࢫࢺࣞࢫฎ⌮ࡢ Col-0 AtALMT1-KO ࡢ 5 ✀࡛࠶ࡿࠋࡑࢀࡒ
ࢀࡢ࣐ࢡࣟࣞᐇ㦂ࡣࠊⰍ⣲ࢆධࢀ᭰࠼ࡓࢲࢫ࣡ࢵࣉࢆྵࡵࠊ⊂❧ࡋࡓ 3
ࡢᐇ㦂ࢆ⾜ࡗ࡚࠸ࡿࠋ࡞࠾ࠊ㔝⏕ᆺࡢCol-0 Col-4 ࡢ Al ప pH ⪏ᛶ⛬ᗘ
ࡣ㠀ᖖఝ࡚࠸ࡿ (Ikka et al., 2007)ࠋ࣐ࢡࣟࣞ㛵ࡍࡿ cDNA ྜᡂࠊcRNA ྜᡂࠊDye ࣛ࣋ࣝࡣ Agilent ࡢ Low RNA Input Linear Amplification Kit PLUS
16
Sequence (5'-) Tm (䉝) Annealing temp. (䉝)
F GGCCGACCGTGCT ATACGAG 62.5
R GAGTT GAAT TACT T ACTGAAG 50.7
F GGCCGACCGTGCT ATACGAG 62.5
R CATGAGT CCT GT GAACT CCC 58.4
F CAGTCGACAT AAGCT GT T TACTT TT 55.6 R GAAT CTCAGCGT CCGACAAT AT AGAA 58.9
F CAT CAGCCAGT ACATCTACTCAGA 58.8
R ATGGCAAT GCCTT AGAGACTAGT A 57.1 F AT GTGTAT GACCGAAAAT GGTACCCGAT C 61.9 R CCT CCATT ACATT GCT CGGGGCT TAAGT 63.4
F T TAATAAACACT GGGAAGACT 50.7
R ACCGCTT T GAGT AGCGT ATACA 56.7
F AAGACAAGAT CCACGAGGACGAC 60.5
R GT CGT AGACATGTT T GGGGGT T G 60.5 F GGT TT ATAACT GAAGCTGTGCTT AG 57.2 R CT ATAACAAAAGT GCCCAAAT AACC 55.6
F AAGGCGATCACGACAGGGGGCGT G 67.3
R TT ATGAGAAACCCCCAAT AGAGATG 57.2 F GTGTT CGT CCT TCAGCGT GT T TACCT GT 63.4 R GCAAGT GGGCT AT TCGTT AACTT CT GTG 62.0
F CACAT ACCGT CT CTACCGTT GA 58.6
R GAGAGAT CT T GGCATGT GT T TGA 56.9 F AGAGAGAT GGCGATAATACAT CAAC 57.2 R AT GGT CCT CCCAAGATT CAT CAT TT 57.2 F T AGTGATCTCT CT GT CTCCGGT T TT 58.8 R GT T TCAAT CT GAGGAGAAGACAT GA 57.2 F TAT CGAT CCT TGCCGGGACTT CA 60.5 R GCT TGT CT TGGCGTT GCT CCTA 60.4 F AGAGAT CCCATT ATCAAACCGAATC 57.2
R CCCAT TAAAACAGTGAAAACGCCTA 57.2
F GCGTAT CAGACGGCT AAGGAGA 60.4
R CCAGGAAT GCTGGAACGACAAAGTT 60.5
F T AGCT GGGAGTT T AGT GACCATAGC 60.5 R AAT TT ATGGAT ACGAT CGAAGCCGA 57.2 F T GT TAGTCCGT GT TGCAGGT GA 58.6 R CACTT CTT GT GCT GAGCCTT GA 58.6 F GCT TT GGACAGCCAGT CGGAACGTG 65.4 R TGAT T ACAAAT AATT T CAT TAGCGA 50.6 F GCGAGAGAGAAAGGGAT GGT TT ATC 60.5 R TCCCCTAAACAT GGAGACACTT T AT 57.2 F ACT TT CAAGCT GGTT GAT CAGAT GG 58.8 R TGGTT AAATACAACT T GCT CT GAAG 55.6 F TAAACCAAGCTT ATCT CT AGGAT TA 53.9 R CCAT CAAAT AAAACATT T TGAAAAT GTGC 54.9
F AGGAGTGACAGT GAGTT ACT TCG 58.7
R TGGCTCGAGCGACT CGGT T A 60.4
F GCACAAGT GACAGAAGAGCAT TAC 58.8
R GAGAGT T CACAAACAGCAT CAT AAT G 57.3 F AGCAT T ACGCCAATGGT CTGATCTA 58.8 R TT CGACGCCAAT CCGAGGT CAT A 60.5
(Table continues on following page.)
At5g11670a 60.0
Table 1. Primer sequences used for quantitative RT-PCR of the markedly down-regulated genes of the
stop1-mutant. Name At1g08430 53.0 At2g37330a 60.0 At2g37330 53.0 At1g34370 53.0 At1g51830 53.0 At1g08430-2 53.0 At1g30270 53.0 60.0 At1g77760 53.0 At2g18480 53.0 At1g51840 53.0 At1g67325 53.0 60.0 At2g37570 53.0 At2g41380 53.0 At2g19900 53.0 At2g28270 53.0 At2g19900a 60.0 At4g13420 53.0 At5g02480 53.0 At3g05400 53.0 At3g28345 53.0 At3g22200a At5g06860 53.0 At5g11670 53.0 At5g07440a
17
Sequence (5'-) Tm (䉝) Annealing temp. (䉝)
F ACAGGAAGAAGACGAGT GGT AT 56.7
R GAAACT TGGT T CAGT AAT AAAGGGA 55.6 F GAAGGT GAACGAT GAGCT AAAGACT 58.8 R GCGTCTT CTAGT ACT T GT AAACACA 57.2
F GTGGCT CAAGCTACCATT CTCAGA 60.5
R CT GT T AACT CAGGAT GACCT CT A 56.9 F CCT TCTT CTT AGT TAT TAGTGGGAA 55.6 R GAAAT T TT CAT TT ATCGCT TCTT GC 53.9 F CGT TT CTT TGT TT T TGGGCCACGTA 58.8 R T CT AT GCATGATACCCAT CAGAACC 58.8 F GGGT TCAAGACGT T GACT T AGTT C 58.8 R TATGGAT CCAAAACAAACCGCT ACT A 57.3 F GCATAT AGAGATCAAAGACAT GAT C 55.6 R GACCT CAT CT AAATT T CCCAT TCCA 57.2 F TGT AT ACAAACACTCACT GAT CATT 53.9 R TCT TT GACGAT AGCAT CAAACTT T C 55.6 F GAGGGATT AT CTT CGATCAAGGCCG 62.1 R CAAGAAGCAAATCTT CCAAT CTCCC 58.8 F GT T TCATGTGAAT GAATCGCATGCA 57.2 R TCATT GTT GGATCTACAT CAGCAAT 55.6 F CGGAT T AGGCCT AAT AAT GGCC 58.6
R AAGAAT TACGATCAACAACGACCAG 57.2
F TCGTAAGT ACAAT CAGGATAAGAT G 55.6
R CACTGAAACAAGAAAAACAAACCCT 55.6
F AGAGCTGTCAACTGCAGGAAGAA 58.7
R ACAAGAAAAACAAACCCT AT CAAAGG 55.8
Table 1. (Continued from previous page.)
Name
At2g37330a primer is used for Real-time RT -PCR in Fig. 3.
At2g19900a, At3g22200a, At5g07440a, At5g11670a, At5g17330, At5g18170a and At3g52590a primer is used for real time RT-PCR in Fig. 6.
At5g18170a 60.0
At1g08430-2 primer is used for RT-PCR in Fig. 4.
At5g17330 53.0 At5g18170 53.0 At5g19600 53.0 At5g22890 53.0 At5g51980 53.0 At5g35410 53.0 At5g27920 53.0 At5g47560 53.0 At3g52590 (internal std.) 53.0
T m indicates melting temparature calculated with GC% method.
At5g38200 53.0
At5g66650 53.0
18
ࢆ⏝ࡋࡓࠋAgilent ࣐ࢡࣟࣞࢫ࢟ࣕࢼ࣮ (G2565BA) ࡛ᚓࡽࢀࡓ⏬ീࡣࠊ
Feature Extraction version 9.5.1 software (Agilent) ࡛ᩘ್ࡋࡓࠋ࣐ࢡࣟࣞ
ࡢࢹ࣮ࢱ࣐ࢽࣥࢢ⤫ィィ⟬ࡣGeneSpring GX version 7.3.1 (Agilent) ࢆ⏝
࠸ࡓࠋ࣐ࢡࣟࣞࢹ࣮ࢱࡢquality control ࡋ࡚ࠊࢫ࣏ࢵࢺࡢᙧ≧ࢹ࣮ࢱࢆ
GeneSpring ࡼ ࡾ flags (low intensity, not uniform, abnormal in the background; http://www.chem.agilent.com/cag/bsp/products/gsgx/downloads/ pdf/FE_Plugin.pdf)ࡋ࡚࣐࣮ࢡࡉࢀࡓࢫ࣏ࢵࢺࢹ࣮ࢱࢆ㝖እࡋࡓࠋ」ᩘࡢฎ⌮༊ (wild type in Al and low-pH treatments ࡲࡓࡣ comparisons between the stop1 mutant and the wild type) ࡛ 2 ࡘ௨ୖࡢࣇࣛࢢࢹ࣮ࢱࡀධࡗ࡚࠸ࡓሙྜࠊࡑࡢ㑇
ఏᏊࡣࢹ࣮ࢱ࣐ࢽࣥࢢࡽ㝖እࡋࡓࠋAlࠊప pH ฎ⌮࠾࠸࡚ࠊstop1 ኚ␗య࡛
㔝⏕ᆺẚ㍑ࡋࡓ㝿ࠊFold Change (FC)࡛࡚ࡢ᳨ฟࡉࢀࡓ㑇ఏᏊࢹ࣮ࢱࡢୗ 1%ྵࡲࢀࡓ㑇ఏᏊࢆࠊⴭࡋࡃ㌿ᢚไࡉࢀࡓ㑇ఏᏊᐃ⩏ࡋࡓࠋ
࣓
࣓ࢱ࣮࣒࣎ࣟゎᯒ
GC-TOF-MS ศᯒࡣࠊLECO Pegasus III mass spectrometer (Agilent) ᥋
⥆ࡉࢀࡓAgilent 6890 GC system ࡛⾜ࡗࡓࠋ୍᪉ࠊCE-MS ศᯒࡣ Agilent 1100
series MSD mass spectrometer ࢆ᥋⥆ࡋࡓࠊAgilent CE system ࡛⾜ࡗࡓࠋCE-MS
ࡼࡿ㝜࢜ࣥࡢ᳨ฟࠊ࠾ࡼࡧGC-TOF-MS ࡼࡿ᳨ฟࡣࠊUrano et al. (2009)
ࡢ᪉ἲᚑࡗࡓࠋ᳨ฟࡉࢀࡓࣆ࣮ࢡࡣࠊ᪤▱ࡢྜ≀(᭷ᶵ㓟ࠊ࣑ࣀ㓟)ࡢ⌮ㄽୖ
ࡢMr್ࠊࡲࡓࡣྜ≀ࡢᶆရࡽ┤᥋᳨ฟࡋࡓ್↷ྜࡋࡓࠋࡑࢀࡒࢀࡢࣆ࣮ࢡ
ࡢᙉᗘࢆfresh weight ࡛ᶆ‽ࡋࡓࠋࡑࢀࡒࢀࡢฎ⌮༊ࢧࣥࣉࣝᑐࡋࠊ⊂❧ࡋ
ࡓ 3 ᅇࡢᐇ㦂ࢆ⾜࠸ࠊExcel ࡛⤫ィゎᯒࢆ⾜ࡗࡓࠋ௦ㅰ≀ࡢẚ㍑್ (: ratio of
normalized peaks of STOP1-KO /wild type, pH 4.5) ࡣ௦ㅰ≀ࡢࣃࢫ࢙࣐࢘ࢵ ࣉ⤫ྜࡋࡓࠋ
19
11-3 ᐇ㦂⤖ᯝ
ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࡼࡿ௦ㅰ㛵㐃㑇ఏᏊࡢAl ᛂ⟅ ᵝࠎ࡞࢜ࣥࢫࢺࣞࢫ࡛ 24 㛫ฎ⌮ࡋࡓ᳜≀యࡢࣜࣥࢦ㓟ྵ㔞ࢆ ᐃࡋࡓ ࡇࢁࠊAl ࡛⣙ 3 ಸࠊAl ࡢ࣑࣑ࢵࢡࡋ࡚ാࡃ LaࠊEr ࡛⣙ 2 ಸቑຍࡋࡓࡀࠊ㏫ పpH ࡸ Naࠊ㔜㔠ᒓฎ⌮࡛ࡣ༙ศ௨ୗῶᑡࡋࡓࠋࡲࡓࠊAl ࡢ⃰ᗘࡀቑ࠼ࡿᚑ ࠸ࣜࣥࢦ㓟ྵ㔞ࡶቑຍࡋ࡚࠸ࡓࡇࡽࠊAl ฎ⌮ࡍࡿࡇ࡛≉␗ⓗࣜࣥࢦ㓟ࡀ ྜᡂࠊ✚ࡍࡿഴྥ࠶ࡿ⪃࠼ࡽࢀࡓࠋࡇࡢ⤖ᯝࡽࣜࣥࢦ㓟ྜᡂ㛵ࢃࡿ㑇ఏ ᏊࡀࠊAl ฎ⌮ࡼࡗ࡚ኚືࡋ࡚࠸ࡿணࡉࢀࡓࡓࡵࠊAl ฎ⌮ࡢ࣐ࢡࣟࣞ ࢹ࣮ࢱࢆ௦ㅰ࣐ࢵࣉ࠶࡚ࡣࡵ࡚௦ㅰ⣔ࡢ㌿ኚືࢆㄪࡓ (Fig. 1a)ࠋ௦ㅰ⣔ య࡛ࡣḟ௦ㅰࠊ◲㯤௦ㅰ㛵ࢃࡿ㑇ఏᏊࡢ㌿㔞ࡀቑຍࡋ࡚࠸ࡓࠋ❅⣲௦ㅰ࣭ ࣑ࣀ㓟௦ㅰ࡛ࡣglutamate dehydrogenase ࢆࡣࡌࡵࡍࡿࢢࣝࢱ࣑ࣥ㓟ࡸࠊࢭࣜ ࣥࠊGABA ࡢྜᡂ⤒㊰࡛㢧ⴭ࡞ቑຍࢆ♧ࡋ࡚࠸ࡓࠋࣜࣥࢦ㓟㛵ࢃࡿ㑇ఏᏊ࡛ࡣࠊTCA ࢧࢡࣝෆࡢ㑇ఏᏊࡣࡸࡸ㌿ࡀᢚ࠼ࡽࢀ࡚࠸ࡿࡢᑐࡋࠊMalic enzymeࠊ Malate dehydrogenaseࠊPEP carboxykinase ࡞ TCA ࢧࢡࣝ࿘㎶ࡢ㑇ఏᏊࡀ
㧗࠸㌿㔞ࢆ♧ࡋ࡚࠸ࡓ (Fig. 1b)ࠋḟࠊAl ࡼࡿ௦ㅰኚືࢆㄪࡓࡇࢁࠊࢢ ࣝࢱ࣑ࣥ㓟ࡀῶᑡࡋࠊࢫࣃࣛࢠࣥ㓟ࡀቑຍࡋ࡚࠸ࡓࠋ௨ୖࡽࠊAl ฎ⌮ࡼࡿ ࣜࣥࢦ㓟ቑຍࡣࢭࣜࣥ⤒㊰ࡸࢢࣝࢱ࣑ࣥ㓟ࡽࢫࣃࣛࢠࣥ㓟ࢆ⤒⏤ࡍࡿ⤒㊰࡞ ࠊTCA ࢧࢡࣝࡢࣂࣃࢫ࡞ࡿ⤒㊰ࡀ㔜せ࡞ᙺࢆࡋ࡚࠸ࡿࡇࡀ♧၀ࡉࢀ ࡓࠋ ࢺࣛࣥࢫࢡࣜࣉࢺ࣮࣒ࡼࡿstop1 ኚ␗య࡛㌿ᢚไࡉࢀࡓ㑇ఏᏊࡢྠᐃ
௨๓ࡢሗ࿌࡛ࡣࠊAl ⪏ᛶ㔜せ࡞㑇ఏᏊࡢ 1 ࡘࡣ AtALMT1 ࡛࠶ࡾࠊAl ฎ⌮
20
F
Figure 1. (a) Metabolism overview by transcriptome in Arabidopsis roots treated by 10 M Al for 24 hours. The color indicated up (red) and down (blue) regulations, or no change (white), compared 10 M Al treated sample for 24 hours with control one. The scale is shown in log2. The result is displayed using the MapMan software. (b) Expression of malate, glutamate and GABA pathway genes in the roots of Arabidopsis thaliana with aluminum treatment. Each transcript was quantified by quantitative RT-PCR with specific primers. Amplicons were stained by SYBR green I. 40S ribosomal are shown as control.
Col-4
GABA-T (AT3G22200) lactate dehydrogenase (AT4G17260)
glutamate decarboxylase (AT5G17330) malate dehydrogenase (AT5G58330)
SSADH1 (AT1G79440) AtNADP-ME2 (AT5G11670) GDH1 (AT5G18170) GDH2 (AT5G07440) 40S ribosomal (AT2G09990) AtNADP-ME1 (AT2G19900)
a
b
21
ࡋࡋࠊAtALMT1 ࡣ H+⪏ᛶࡣ㈉⊩ࡋ࡚࠸࡞ࡗࡓ (Kobayashi et al., 2007)ࠋ
ࡇࡢ⤖ᯝࡣࠊSTOP1 ࡀ AtALMT1 ௨እࡢ Al H+⪏ᛶ㑇ఏᏊࡢ㌿ㄪ⠇ࢩࢢࢼࣝ
ఏ㐩㛵ࢃࡗ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋࡇࢀࢆㄪࡿࡓࡵࠊࡲࡓࡑࡢ㑇ఏᏊࢆ≉
ᐃࡍࡿࡓࡵࠊ㑇ఏᏊࡢ㌿㔞ࢆ⥙⨶ⓗㄪࡿDNA ࣐ࢡࣟࣞࢆ⾜ࡗࡓࠋ᭱
ึࠊAlࠊH+ࢫࢺࣞࢫࢆཷࡅࡓ㔝⏕ᆺstop1 ኚ␗య࡛㌿㔞ࢆẚ㍑ࡋࠊstop1 ኚ
␗య࡛ࠊࡼࡾ㌿ᢚไࡉࢀࡓ㑇ఏᏊࢆỴᐃࡍࡿࡓࡵࠊ㌿㔞ࢆẚ㍑ࡍࡿ࣐ࢡࣟ
ࣞࢆ⾜ࡗࡓࠋ⊂❧ࡋࡓ3 ᅇࡢᐇ㦂ࢆ⾜࠸ࠊAlࠊH+ฎ⌮ࡑࢀࡒࢀ࠾࠸࡚ࠊstop1
ኚ␗య࡛ⴭࡋࡃ㌿ᢚไࡉࢀࡓ㑇ఏᏊࢆfold change (FC)್ࠊ(stop1 ኚ␗య / 㔝
⏕ᆺ) ࡛㑅ᢤࢆ⾜ࡗࡓࠋ3 ᅇࡢᐇ㦂ࡍ࡚࠾࠸࡚ୗ 1%ྵࡲࢀࡓ㑇ఏᏊࡣ ⌧ྍ⬟࡞㌿ᢚไ㑇ఏᏊࡋࠊAl ࡛ࡣ 101ࠊH+࡛ࡣ45 ࡢ㑇ఏᏊࡀ㑅ᢤࡉࢀࡓ (Fig. 2a)ࠋࡇࢀࡽࡢ㑇ఏᏊࡢ࠺ࡕࠊAl H+ฎ⌮ࡢ୧᪉࡛㌿ᢚไࡉࢀࡓࡢࡣࠊ32 㑇ఏ Ꮚ࡛࠶ࡗࡓࠋ ࡇࡇ࡛ࠊstop1 ኚ␗య࡛㌿ᢚไࡉࢀࡓ㑇ఏᏊࡢ୰ࡣࠊᡂ㛗ࡀ㔝⏕ᆺࡼࡾࡶ ᖜ㜼ᐖࡉࢀࡓࡇࡼࡾ㌿ᢚไࡉࢀࡓࠊᡂ㛗㛵㐃ࡍࡿ㑇ఏᏊࡀྵࡲࢀ࡚࠸ ࡿྍ⬟ᛶࡀ࠶ࡿࠋࡇࡢࡼ࠺࡞㑇ఏᏊࡣࠊAl ࡸ H+ฎ⌮ࡼࡗ࡚㌿ᢚไࡉࢀࡿࡇ ࡛⏕⫱ࡀᝏࡃ࡞ࡿ⪃࠼ࡽࢀࡿࠋࡑࡇ࡛ࠊAl ฎ⌮ / control ࡸ H+ฎ⌮ / control ࡢ 㔝⏕ᆺࡢࢫࢺࣞࢫㄏᑟ࣐ࢡࣟࣞࢆ⾜࠸ࠊFC ್࡛ࡑࡢࡼ࠺࡞㑇ఏᏊࢆ㑅ᢤࡋ ࡓࠋ㑅ᢤࡢຠᯝࢆ᭱㝈ࡍࡿࡓࡵࠊฎ⌮༊࠾࠸࡚᭷ព㌿ᢚไࡉࢀࡓ㑇ఏ Ꮚ (FC <1ࠊࡘt-test ࡛ P <0.05)ࠊFC ࡀ 0.8 ௨ୗࡢ㑇ఏᏊࢆ㝖࠸ࡓࠋࡇࡢ⤖ ᯝࠊAl ࡛ 64 ಶࠊH+࡛28 ಶ (㔜」 19 ಶࢆྵࡴ)ࡢ㑇ఏᏊࡀࣜࢫࢺࡽ㝖ࢀࡓ (Fig. 2aࠊTable 2)ࠋࡇࡢࡼ࠺ࡋ࡚ṧࡗࡓ㑇ఏᏊࢆࠊstop1 ኚ␗య࡛᭷ព㌿ᢚไࡉ ࢀࡓ㑇ఏᏊࡋࡓࠋࡑࡢ༙ศ௨ୖࡣࠊAtALMT1 ࡢ T-DNA ᤄධࣀࢵࢡ࢘ࢺࣛ ࣥ (AtALMT1-KO)ࡢ Al ฎ⌮࡛㌿ᢚไ (FC [AtALMT1-KO / 㔝⏕ᆺ] <1ࠊࡘ t-test ࡛ P <0.05 ࡲࡓࡣ FC <0.8) ࡉࢀ࡚࠾ࡾ(Table 2ࠊTable 3)ࠊ
22
Figure 2. (a) Genes repressed in the stop1 mutant compared with the wild type (WT) that were identified by competitive microarray analyses. Greatly repressed genes in the stop1 mutant, when they were compared with the wild type, were identified by three biologically independent replications of the comparative microarray of the stop1 mutant/wild type under Al toxic (10 M) and low-pH (pH 4.5) conditions. The genes that were in the lowest percentile of the FC values (stop1 mutant/wild type) in all three replications were defined as ‘‘significantly down-regulated genes.’’ Numbers of down-regulated genes in the wild type in each treatment (FC in treatments/control in the wild type were < 1 with P < 0.05 and FC < 0.8) are shown in the lower parts of the Venn diagram. The genes down-regulated in AtALMT1-KO compared with the wild type in the upper parts are shown by italics. (b) Expression levels of 12 overlapped genes in the wild type, the stop1 mutant, STOP1-KO, and a complemented stop1 mutant carrying CaMV35S::STOP1 (stop1-comp) were determined by semiquantitative RT-PCR.
23 F C ± S D P -v al ue F C ± S D P -v al ue F C ± S D P -v al ue F C ± S D P -v al ue F C ± S D P-va lu e A t1 g5183 0 0. 02 ± 0. 00 0.0 01 0.1 0 ± 0.0 3 0.00 7 0.98 ± 0.14 0.83 0 2.6 5 ± 0.7 5 0.0 26 0. 63 ± 0 .22 0. 14 7 A T P bi ndi ng / ki na se / p ro te in s er in e/ th re oni ne k in as e A t1 g5185 0 0. 10 ± 0. 01 0.0 00 0.4 3 ± 0.0 7 0.01 4 0.87 ± 0.12 0.22 6 0.4 5 ± 0.1 6 0.0 54 0. 38 ± 0 .05 0. 00 5 p ut at iv e, l eu ci ne -r ic h re p ea t p ro te in ki na se A t1 g5207 0 0. 01 ± 0. 00 0.0 00 0.0 3 ± 0.0 2 0.01 3 1.04 ± 0.15 0.66 8 0.4 0 ± 0.2 1 0.0 83 0. 05 ± 0 .01 0. 00 1 ja ca lin le ct in fa m ily p ro te in A t1 g5643 0 0. 31 ± 0. 02 0.0 01 0.3 7 ± 0.1 6 0.05 3 2.01 ± 0.32 0.01 6 0.1 6 ± 0.0 1 0.0 01 1. 07 ± 0 .10 0. 33 8 N A S4 ( ni co ti an am ine s y nt ha se 4) A t1 g6732 5 0. 24 ± 0. 02 0.0 01 0.2 5 ± 0.0 1 0.00 1 1.00 ± 0.14 0.97 7 0.9 3 ± 0.0 8 0.2 53 0. 80 ± 0 .13 0. 14 0 R an BP 2-ty p e z in c fi ng er p rot ei n A t1 g8024 0 0. 06 ± 0. 01 0.0 01 0.3 0 ± 0.0 9 0.02 1 0.95 ± 0.13 0.56 9 0.8 3 ± 0.0 9 0.0 86 0. 32 ± 0 .05 0. 00 7 unk now n p ro te in A t2 g4348 0 0. 31 ± 0. 05 0.0 07 0.3 9 ± 0.0 9 0.01 8 1.03 ± 0.15 0.79 8 0.7 2 ± 0.1 7 0.1 39 0. 51 ± 0 .18 0. 07 4 P E R 26 ( p er ox ida se 2 6 p re cu rs or ) A t2 g4411 0 0. 29 ± 0. 02 0.0 01 0.3 8 ± 0.0 2 0.00 1 0.98 ± 0.34 0.91 5 0.3 8 ± 0.0 5 0.0 07 0. 35 ± 0 .02 0. 00 1 M L O 15 ( m ilde w r es is ta nc e loc us o 15) A t3 g2038 0 0. 08 ± 0. 04 0.0 11 0.2 9 ± 0.1 1 0.02 8 1.23 ± 0.18 0.12 9 0.8 5 ± 0.0 9 0.1 09 0. 58 ± 0 .30 0. 19 3 M A T H dom ai n-c ont ai ni ng p ro te in A t3 g2834 5 0. 15 ± 0. 01 0.0 00 0.2 6 ± 0.0 5 0.00 6 1.24 ± 0.18 0.12 3 0.6 5 ± 0.4 0 0.3 12 1. 29 ± 0 .24 0. 13 9 M D R 13 (A BC t ra ns p or te r fa m ily p ro te in ) A t3 g4627 0 0. 12 ± 0. 02 0.0 01 0.2 8 ± 0.0 4 0.00 4 0.73 ± 0.18 0.08 9 0.3 5 ± 0 .0 3 0. 00 2 0. 49 ± 0 .1 9 0. 07 8 recep to r p ro tein k in as e-relat ed A t3 g4628 0 0. 08 ± 0. 02 0.0 03 0.2 5 ± 0.0 6 0.00 8 0.79 ± 0.12 0.11 1 0.3 4 ± 0 .0 7 0. 01 0 0. 29 ± 0 .0 4 0. 00 4 recep to r p ro tein k in as e-relat ed A t3 g5064 0 0. 18 ± 0. 03 0.0 03 0.4 1 ± 0.0 5 0.00 6 0.50 ± 0.08 0.01 6 0.3 1 ± 0.0 7 0.0 12 0. 15 ± 0 .03 0. 00 5 unk now n p ro te in A t3 g6194 0 0. 21 ± 0. 05 0.0 07 0.2 4 ± 0.1 2 0.03 3 2.90 ± 0.76 0.00 2 0.0 4 ± 0.0 0 0.0 00 0. 36 ± 0 .03 0. 00 3 A T M T P A 1 (z inc t ra ns p or te r fa m ily ) A t4 g3067 0 0. 11 ± 0. 01 0.0 01 0.3 1 ± 0.0 7 0.01 2 1.15 ± 0.16 0.23 3 2.2 0 ± 0.4 2 0.0 19 0. 52 ± 0 .04 0. 00 4 unk now n p ro te in A t5 g0386 0 0. 29 ± 0. 06 0.0 10 0.4 2 ± 0.0 5 0.00 5 2.09 ± 0.52 0.00 7 0.3 9 ± 0.0 5 0.0 06 0. 69 ± 0 .10 0. 04 6 M al at e sy nt ha se A t5 g2546 0 0. 03 ± 0. 00 0.0 01 0.2 2 ± 0.1 1 0.03 0 0.92 ± 0.13 0.39 1 2.8 4 ± 0.5 3 0.0 10 0. 30 ± 0 .05 0. 00 6 unk now n p ro te in A t5 g2792 0 0. 15 ± 0. 02 0.0 02 0.1 5 ± 0.0 0 0.00 0 0.99 ± 0.18 0.95 0 1.2 6 ± 0.2 3 0.1 61 0. 90 ± 0 .20 0. 50 4 si m ila r t o F B L 3 ( F -box f am ily p rot ei n) A t5 g5878 4 0. 12 ± 0. 02 0.0 02 0.2 9 ± 0.1 2 0.03 2 0.46 ± 0.08 0.01 5 0.6 0 ± 0.1 9 0.1 03 0. 39 ± 0 .05 0. 00 7 de hy dr odo lic hy l d ip ho sp ha te s y nt ha se 5 A t1 g0175 0 0. 15 ± 0. 02 0.0 01 0.4 5 ± 0.2 1 0.08 8 0.33 ± 0.07 0.00 4 0.2 9 ± 0.0 6 0.0 10 0. 27 ± 0 .06 0. 01 0 A D F 10 ( ac ti n-de p ol y m er iz in g fa ct or 10 ) A t1 g0525 0 0. 12 ± 0. 08 0.0 25 0.5 3 ± 0.2 3 0.11 4 1.54 ± 0.23 0.03 7 0.1 4 ± 0.0 1 0.0 01 0. 08 ± 0 .01 0. 00 1 P E R 2 (p er ox id as e 1/ 2 p re cu rs or) A t1 g1209 0 0. 28 ± 0. 02 0.0 01 0.8 0 ± 0.1 0 0.08 9 0.50 ± 0.08 0.01 7 0.3 3 ± 0.1 1 0.0 26 0. 25 ± 0 .05 0. 00 7 E L P ( ex te ns in -l ik e p rot ei n) A t1 g1422 0 0. 20 ± 0. 07 0.0 14 0.6 6 ± 0.2 1 0.14 7 0.83 ± 0.13 0.17 1 0.3 9 ± 0.0 3 0.0 02 0. 27 ± 0 .03 0. 00 3 si m ila r t o RN S3 ( ri bon uc le as e 3) A t1 g2250 0 0. 15 ± 0. 04 0.0 06 0.9 6 ± 0.1 8 0.72 0 0.99 ± 0.14 0.92 4 0.3 3 ± 0.1 2 0.0 34 0. 68 ± 0 .29 0. 24 0 A T L 1C (RI N G -H 2 f ing er p ro te in A T L 1C p recu rs or ) A t1 g2375 0 0. 23 ± 0. 03 0.0 03 0.5 1 ± 0.1 6 0.05 9 0.51 ± 0.08 0.01 8 0.4 0 ± 0.1 3 0.0 37 0. 26 ± 0 .08 0. 01 6 D N A -b in di ng p ro te in -r el at ed A t1 g2714 0 0. 07 ± 0. 03 0.0 08 0.4 9 ± 0.1 0 0.02 4 0.21 ± 0.07 0.00 1 0.5 3 ± 0.2 4 0.1 27 0. 24 ± 0 .06 0. 01 1 A T G ST U 14 ( gl ut at hi one S䇲t ran sf er as e T A U 1 4) A t1 g3087 0 0. 08 ± 0. 03 0.0 07 0.6 8 ± 0.1 5 0.08 8 0.30 ± 0.07 0.00 1 0.1 0 ± 0 .0 2 0. 00 2 0. 10 ± 0. 02 0. 00 3 P ER 7 (p er ox id as e 7 p rec ur so r) A t1 g4893 0 0. 15 ± 0. 01 0.0 00 0.7 6 ± 0.1 2 0.09 6 0.42 ± 0.09 0.00 3 0.1 6 ± 0.0 3 0.0 03 0. 17 ± 0 .04 0. 00 6 A T G H 9C1 ( gl y co sy l h y dr ol as e 9C1) A t1 g4903 0 0. 11 ± 0. 04 0.0 09 0.5 0 ± 0.1 7 0.06 9 0.24 ± 0.08 0.00 2 0.4 8 ± 0.0 4 0.0 04 0. 54 ± 0 .10 0. 02 9 unk now n p ro te in A t1 g4986 0 0. 29 ± 0. 03 0.0 02 0.6 3 ± 0.2 6 0.18 0 1.06 ± 0.30 0.73 8 0.2 9 ± 0.1 4 0.0 41 0. 85 ± 0 .24 0. 41 4 A T G ST F 14 (g lu ta th ione t ra ns fe ra se ) A t1 g6647 0 0. 30 ± 0. 02 0.0 01 0.8 3 ± 0.1 0 0.11 7 0.71 ± 0.10 0.05 6 0.8 2 ± 0.2 1 0.3 10 0. 55 ± 0 .07 0. 01 3 ba si c he lix -l oo p -he lix (bH L H ) fa m ily pr ot ei n A t1 g7578 0 0. 30 ± 0. 03 0.0 02 0.6 7 ± 0.0 6 0.01 4 0.72 ± 0.11 0.05 9 0.4 9 ± 0.2 4 0.1 18 0. 24 ± 0 .06 0. 00 9 T U B 1 (T ub ul in b et a-1 ch ain 䠅 A t2 g0416 0 0. 21 ± 0. 03 0.0 03 0.5 4 ± 0.1 1 0.03 6 1.30 ± 0.19 0.08 7 0.4 3 ± 0.2 1 0.0 88 0. 17 ± 0 .04 0. 00 6 A IR 3 ( aux in -i nd uc ed in ro ot c ul tu re s 3) A t2 g2581 0 0. 13 ± 0. 04 0.0 06 0.6 8 ± 0.1 5 0.09 5 0.28 ± 0.06 0.00 5 0.5 1 ± 0.0 4 0.0 05 0. 59 ± 0 .15 0. 06 9 T IP 4; 1 ( ton op la st in tr ins ic p ro te in 4 ;1 ) A t2 g2737 0 0. 28 ± 0. 07 0.0 12 0.6 2 ± 0.1 1 0.04 6 0.75 ± 0.11 0.07 2 0.6 3 ± 0.0 0 0.0 00 0. 60 ± 0 .09 0. 02 9 int eg ra l m em bra ne f am ily p ro te in A t2 g3418 0 0. 25 ± 0. 04 0.0 04 1.4 5 ± 0.4 5 0.17 1 1.54 ± 0.30 0.01 9 0.6 0 ± 0.0 5 0.0 09 0. 78 ± 0 .31 0. 38 4 C IP K 13 ( CBL -i nt er ac ti ng p ro te in ki na se 13) A t2 g3943 0 0. 32 ± 0. 04 0.0 05 0.5 7 ± 0.1 4 0.05 9 0.79 ± 0.12 0.08 5 0.2 5 ± 0.0 8 0.0 16 0. 17 ± 0 .01 0. 00 0 di se as e re si st an ce -re sp ons iv e p rot ei n-relat ed A t3 g1071 0 0. 27 ± 0. 12 0.0 34 0.7 5 ± 0.2 6 0.27 5 1.50 ± 0.24 0.04 6 0.2 5 ± 0.1 0 0.0 24 0. 29 ± 0 .13 0. 03 6 p ec ti ne st er as e fa m ily p ro te in A t3 g4396 0 0. 27 ± 0. 05 0.0 07 0.6 0 ± 0.0 8 0.02 1 0.64 ± 0.09 0.03 3 0.5 1 ± 0.1 0 0.0 26 0. 34 ± 0 .04 0. 00 4 p ut at iv e cy st ei ne p ro te in as e A t3 g5937 0 0. 17 ± 0. 04 0.0 06 0.4 6 ± 0.0 9 0.01 8 0.24 ± 0.06 0.00 8 0.4 8 ± 0.2 0 0.0 85 0. 36 ± 0 .11 0. 02 7 unk now n p ro te in (T ab le c ont inue s o n f ol lowi ng pa ge .) lo w p H s top1 -m ut an t A l At AL M T -K O Al lo w p H Tab le 2 . L is t o f d own-re gul at ed ge ne s i n t he s to p1-m ut ant a m on g do w n-re gul at ed o r s ta bl e ge ne s i n W T und er A l and lo w pH c on di ti ons . R ep re sse d in st op 1 -m ut an t, a nd d ow n-r eg ul at ed or s ta bl e in W T w it h A l a nd low p H t re at m ent s; 19 ge ne s R ep re sse d in st op 1 -m ut an t, a nd d ow n-reg ul at ed o r st ab le in W T w it h A l t reat m en t; 45 ge ne s AG I co de Fo ld c ha ng e (st op 1 -m ut an t or At A LM T1 -KO/ W T ) Fo ld ch an ge in t he W T w it h Al o r lo w p H t reat m en ts ( tr ea tm en t/ co nt ro l) De sc ri p ti on Al s top1 -m ut an t
24 F C ± S D P -v al ue F C ± S D P -v al ue F C ± S D P -v al ue F C ± S D P -v al ue F C ± S D P-va lu e Re p re ss ed in st op 1 -m ut an t, a nd dow n-re gu la te d or s ta bl e in W T w it h A l t re at m en t; 45 g ene s ( C ont inue d f rom pr ev ious page .) A t4g 0008 0 0.1 6 ± 0.03 0.002 0.68 ± 0.18 0.1 27 0.91 ± 0. 13 0.345 0.73 ± 0 .0 4 0. 00 9 0. 42 ± 0 .0 7 0. 01 2 U N E1 1 (u nf er tiliz ed e mb ry o sa c 11 ) A t4g 0195 0 0.2 2 ± 0.03 0.0 02 0.68 ± 0.19 0.139 0.53 ± 0. 08 0.019 0.60 ± 0.04 0.006 0.69 ± 0 .02 0.001 A T G P A T 3 ( gl y ce ro l- 3-p ho sp ha te a cy lt ra ns fe ra se 3 ) A t4g 1035 0 0.3 2 ± 0.02 0.0 01 0.49 ± 0.17 0.066 0.89 ± 0. 18 0.376 0.37 ± 0.04 0.003 0.27 ± 0 .02 0.002 A N A C 070 ( N A C dom ai n c ont ai ni ng p ro te in 70) A t4g 1903 0 0.2 5 ± 0.05 0.0 08 0.59 ± 0.03 0.003 0.57 ± 0. 09 0.023 0.34 ± 0.10 0.024 0.16 ± 0 .02 0.001 N IP 1; 1 ( N O D 26-lik e in tr in si c p rot ei n 1; 1) A t4g 2556 0 0.3 1 ± 0.03 0.0 03 0.73 ± 0.09 0.048 0.91 ± 0. 13 0.365 0.58 ± 0.08 0.020 0.44 ± 0 .02 0.002 A T M Y B 18 ( M Y B dom ai n p ro te in 18) A t4g 2601 0 0.1 0 ± 0.05 0.0 14 0.86 ± 0.29 0.501 0.79 ± 0. 14 0.087 0.14 ± 0.04 0.008 0.05 ± 0 .02 0.004 P E R 44 ( p er ox id as e 44 p re cu rs or ) A t4g 3147 0 0.1 8 ± 0.05 0.0 08 0.59 ± 0.11 0.041 0.41 ± 0. 08 0.004 0.20 ± 0.02 0.002 0.17 ± 0 .04 0.005 p ut at ive p at hog en es is -r el at ed p ro te in A t4g 3194 0 0.1 3 ± 0.09 0.0 32 0.34 ± 0.25 0.105 1.09 ± 0. 35 0.654 0.04 ± 0.01 0.005 0.21 ± 0 .27 0.113 C Y P 82C 4 ( cy toc hr om e P 450, f am ily 82, subf am ily C , p ol y p ep ti de 4) A t4g 3388 0 0.1 3 ± 0.04 0.0 08 0.51 ± 0.07 0.013 0.56 ± 0. 10 0.028 0.18 ± 0.03 0.004 0.37 ± 0 .10 0.022 ba si c he lix -l oop -h el ix ( bH L H ) fa m ily pr ot ei n A t4g 3458 0 0.1 6 ± 0.04 0.0 07 0.59 ± 0.14 0.056 0.83 ± 0. 13 0.178 0.13 ± 0.02 0.001 0.09 ± 0 .04 0.009 si m ila r to S E C 14 c y to so lic f ac to r fa m ily p ro te in A t4g 3493 0 0.3 4 ± 0.01 0.0 00 0.67 ± 0.15 0.087 0.71 ± 0. 11 0.049 0.72 ± 0.10 0.060 1.12 ± 0 .15 0.295 1-p hos p ha ti dy linos it ol p hos p hodi es te ras e-r el at ed A t4g 3716 0 0.1 7 ± 0.02 0.0 01 0.41 ± 0.13 0.035 0.48 ± 0. 08 0.017 0.37 ± 0.21 0.085 0.15 ± 0 .06 0.015 SK S15 ( SK U 5 S im ila r 15 ) A t5g 0550 0 0.3 2 ± 0.03 0.0 02 0.82 ± 0.11 0.117 0.50 ± 0. 08 0.017 0.19 ± 0.04 0.005 0.28 ± 0 .06 0.008 p ol le n O le e 1 al le rg en a nd e xt ens in f amily p ro te in A t5g 1294 0 0.3 2 ± 0.01 0.0 00 0.50 ± 0.10 0.027 1.05 ± 0. 15 0.619 0.80 ± 0.22 0.285 0.34 ± 0 .06 0.008 le uc in e-ri ch r ep ea t fa m ily p ro te in A t5g 1515 0 0.2 1 ± 0.05 0.0 08 0.72 ± 0.08 0.033 0.73 ± 0. 19 0.096 0.29 ± 0.08 0.016 0.31 ± 0 .24 0.099 A T H B -3 ( ar ab id op si s th al ia na hom eobox 3) A t5g 2407 0 0.2 1 ± 0.03 0.0 02 0.48 ± 0.16 0.061 0.61 ± 0. 22 0.096 0.10 ± 0.03 0.004 0.05 ± 0 .01 0.002 P E R 61 ( p er ox id as e 61 p re cu rs or ) A t5g 2410 0 0.2 6 ± 0.06 0.0 11 0.60 ± 0.08 0.022 1.07 ± 0. 15 0.517 0.49 ± 0.08 0.015 0.25 ± 0 .08 0.016 le uc in e-ri ch r ep ea t tr an sm em br an e p ro te in k in as e A t5g 4218 0 0.2 8 ± 0.05 0.0 07 0.62 ± 0.08 0.024 0.90 ± 0. 13 0.308 0.27 ± 0.08 0.014 0.18 ± 0 .05 0.007 P E R 64 ( p er ox id as e 64 p re cu rs or ) A t5g 4402 0 0.3 1 ± 0.05 0.0 05 1.04 ± 0.14 0.668 1.07 ± 0. 15 0.513 0.22 ± 0.11 0.029 0.14 ± 0 .02 0.002 ac id p ho sp ha ta se c la ss B f am ily p ro te in A t5g 4927 0 0.1 8 ± 0.03 0.0 04 0.63 ± 0.18 0.107 0.39 ± 0. 07 0.010 0.44 ± 0.17 0.060 0.37 ± 0 .10 0.023 C O B L 9 (c obr a-lik e p rot ei n 9) A t5g 5325 0 0.2 6 ± 0.03 0.0 03 0.63 ± 0.18 0.106 0.79 ± 0. 11 0.104 0.28 ± 0.03 0.002 0.35 ± 0 .09 0.018 A G P 22 ( A ra bi nog al ac ta n p ep ti de 22 p re cu rs or ) A t5g 5632 0 0.2 3 ± 0.03 0.0 02 0.65 ± 0.06 0.014 0.74 ± 0. 11 0.071 0.54 ± 0.14 0.056 0.29 ± 0 .02 0.001 A T E X P A 14 ( ar abi dop si s th al ia na e xp an si n A 14) A t5g 5762 5 0.1 5 ± 0.07 0.0 17 0.57 ± 0.11 0.038 0.72 ± 0. 13 0.047 0.09 ± 0.01 0.000 0.11 ± 0 .06 0.015 al le rg en V 5/ T p x- 1-re la te d f am ily p ro tei n A t5g 6053 0 0.3 1 ± 0.02 0.0 01 0.90 ± 0.08 0.159 0.51 ± 0. 10 0.005 0.10 ± 0.06 0.017 0.06 ± 0 .01 0.000 la te e m br y og en es is a bunda nt p ro te in -r el at ed A t1g 2817 0 0.2 6 ± 0.18 0.0 64 0.41 ± 0.11 0.029 0.88 ± 0. 17 0.315 0.67 ± 0.42 0.350 0.49 ± 0 .31 0.168 su lf ot ra ns fe ra se f am ily p ro te in A t1g 3293 0 0.4 2 ± 0.08 0.0 15 0.40 ± 0.06 0.010 0.98 ± 0. 16 0.868 0.91 ± 0.06 0.125 0.81 ± 0 .04 0.014 ga la ct os y lt ra ns fe ra se f am ily p ro te in A t1g 4970 0 0.4 6 ± 0.06 0.0 09 0.29 ± 0.09 0.020 0.78 ± 0. 37 0.427 0.45 ± 0.21 0.087 0.68 ± 0 .19 0.136 un know n p rot ei n A t1g 8083 0 0.3 1 ± 0.09 0.0 19 0.41 ± 0.14 0.043 1.09 ± 0. 16 0.402 0.36 ± 0.14 0.043 0.68 ± 0 .28 0.231 N R A M P 1 ( na tu ra l r es is ta nc e-as so ci at ed m ac ro p hag e p ro tei n 1) A t2g 3782 0 0.2 9 ± 0.10 0.0 23 0.33 ± 0.06 0.009 0.66 ± 0. 11 0.048 0.18 ± 0.03 0.004 0.54 ± 0 .12 0.038 D C 1 dom ai n-cont ai ni ng p ro te in A t3g 4736 0 0.3 6 ± 0.03 0.0 03 0.34 ± 0.12 0.030 0.96 ± 0. 14 0.693 0.68 ± 0.08 0.029 0.87 ± 0 .06 0.082 A T H SE 3 ( hy dr ox y st er oi d de hy dr og en as e 3) A t5g 0957 0 0.9 7 ± 0.03 0.1 53 0.42 ± 0.13 0.037 1.01 ± 0. 14 0.947 0.36 ± 0.11 0.029 0.88 ± 0 .22 0.472 un know n p rot ei n A t5g 1474 0 0.3 2 ± 0.07 0.0 10 0.35 ± 0.15 0.048 2.45 ± 0. 67 0.006 0.27 ± 0.11 0.028 0.73 ± 0 .04 0.012 C A 1 ( ca rboni c anhy dr as e 1) A t5g 5198 0 0.3 4 ± 0.01 0.0 00 0.32 ± 0.03 0.002 1.02 ± 0. 14 0.866 0.98 ± 0.10 0.734 0.91 ± 0 .06 0.146 Z F W D 2 (W D -40 r ep ea t f am ily p ro te in ) lo w p H s top1 -m ut an t A l At A LM T -K O Al lo w p H Tab le 2 . (C ont inue d f ro m p re vi ous pag e. ) A G I code Fo ld c ha ng e (s top1 -mu ta nt o r At A L M T1 -K O /W T ) Fol d ch an ge in th e W T w it h A l o r lo w pH t re at m ent s (t re at m en t/ co nt ro l) De sc ri p ti on Al s top1 -m ut an t Re p re ss ed in st op 1 -m ut an t, a nd dow n-re gu la te d or s ta bl e in W T w it h l ow p H t re at m ent ; 9 g ene s B io lo gic ally in de p en de nt 3 re p lic at io n of co m p et it iv e mic ro ar ra y an al y se s w er e car ri ed ou t as fo llo w s; st op 1-m ut an t ve rs us w ild ty p e (W T ) in A l(1 0 μM ,24 h) or lo w p H (p H 4. 5, 24 h) ; At AL M T 1-K O ve rs us W T in A l( 10 μM , 24 h) ; C on tr ol (p H 5.0 no A l, 24 h) ve rs us A l ( 10 μ M , p H 5. 0, 24 h) or lo w p H ( p H 4. 5, 24 h) in W T . F ol d c ha ng e (F C ) a nd w hos e ± S D a re s ho w n. P -v al ue of t -t es t f or s ig ni fi ca nt di ff er enc e fr om f ol d ch an ge 1.00 i s show n.
25 AtALMT1-KO ࡢᡂ㛗ࡣ㔝⏕ᆺẚ㜼ᐖࡉࢀ࡚࠸ࡓࠋࡇࡢ⤖ᯝࡣࠊ๓㏙ࡢ㑅ᢤ᪉ ἲࡀࠊstop1 ኚ␗యࡢࡼ࠺࡞ឤཷᛶಶయࡢᡂ㛗ᢚไ⏤᮶ࡍࡿഇ㝧ᛶࡢ㝖ཤ᭷ຠ ࡛࠶ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋࡇࢁ࡛ࠊAl ฎ⌮ప pH ฎ⌮ࡢ୧᪉ศ㢮ࡉࢀࡓࠊ 㔝⏕ᆺࡢ Al ࢫࢺࣞࢫㄏᑟ࣐ࢡ࡛ࣟࣞ㌿ᢚไࡉࢀࡎࠊࡘ AtALMT1-KO ࡛᭷ព㌿ᢚไࡉࢀࡓ㑇ఏᏊࡀ1 ࡘࡔࡅ࠶ࡗࡓࠋࡇࡢ㑇ఏᏊࠊAt1g53480 ࡣstop1 ኚ␗య࡛ࡣ Al ㄏᑟᛶࡢࣜࣥࢦ㓟ᨺฟࡀ㜼ᐖࡉࢀࡓࡓࡵ㌿ᢚไࡉࢀࡓࡀࠊ STOP1 ࡢኚ␗ࡑࡢࡶࡢࡼࡿ⤖ᯝ࡛ࡣ࡞࠸ྍ⬟ᛶࡀ࠶ࡿࠋ᭱⤊ⓗࠊ12 ಶࡢ Alࠊ పpH ඹ㏻㑇ఏᏊࢆྵࡴࠊྜィ 39 ಶࡢ㌿ᢚไ㑇ఏᏊࢆ㑅ᢤࡋࡓ (Table 3ࠊFig. 2a)ࠋ stop1 ኚ␗య࠾ࡅࡿ㑇ఏᏊࡢᢚไࡀࠊSTOP1 ࡢኚ␗ࡼࡗ࡚㉳ࡇࡗࡓྍ⬟ᛶ ࢆࡉࡽ᳨ドࡍࡿࡓࡵࠊඹ㏻ࡍࡿ12 ಶࡢ㑇ఏᏊࡢ Alࠊప pH ฎ⌮ࡢ㌿ࣞ࣋ࣝ
ࢆࠊSTOP1-KO (STOP1 ࡢ T-DNA ᤄධኚ␗య)ࠊ┦⿵ࣛࣥ (࢝ࣜࣇ࣮ࣛ࣡
ࣔࢨࢡ࢘ࣝࢫ (35S CaMV)ࣉ࣮ࣟࣔࢱ࣮ STOP1 㑇ఏᏊࢆ᥋⥆ࡋࡓ࣋ࢡࢱ
࣮ࢆࠊstop1 ኚ␗యᑟධࡋࡓ⤌య (Iuchi et al., 2007))࡛ẚ㍑ࡋࡓࠋࡑࡢ⤖ᯝࠊ stop1 ኚ␗య STOP1-KO ࡢ㌿ࣃࢱ࣮ࣥࡣ㠀ᖖఝ࡚࠸ࡓ (Fig. 2b)ࠋࡉࡽࠊ
STOP1 ࢆstop1 ኚ␗యᑟධࡋࡓ┦⿵࡛ࣛࣥࡣࠊࡇࢀࡽ 12 ಶࡢ㑇ఏᏊࡢ㌿
㔞ࡀᅇࡋࡓ (Fig. 2b)ࠋ௨ୖࡢ⤖ᯝࡣࠊSTOP1 ࡢኚ␗ࡀࠊࡇࢀࡽࡢ㑅ᢤ㑇ఏᏊࡢ ㌿ᙳ㡪ࢆ࠼࡚࠸ࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋ
sstop1 ኚ␗య࠾ࡅࡿ㑇ఏᏊᢚไࡢᶵ⬟
stop1 ኚ␗య࠾࠸࡚ Al ప pH ฎ⌮ࡢ୧᪉࡛㌿ᢚไࡉࢀࡿඹ㏻ࡢ㑇ఏᏊࢢ
࣮ࣝࣉࡣࠊ㐣ཤ༢㞳ࡉࢀࡓAl ⪏ᛶ㔜せ࡞ AtALMT1 (Hoekenga et al., 2006;
Table 3)ࡸࠊzinc finger protein ࢆࢥ࣮ࢻࡍࡿ STOP1 ࡢ࣍ࣔࣟࢢ㑇ఏᏊ (௨ୗࠊ S T O P 2 ) ࡀ ྵ ࡲ ࢀ ࡚࠸ ࡓ ࠋ ࠊ ◲ 㓟 ࢺࣛ ࣥ ࢫ ࣏ ࣮ ࢱ ࣮ ࡢ S U LT R 3 ; 5 ࠊ
26
FC ± SD P -value FC ± SD P -value FC ± SD P -value FC ± SD P -value FC ± SD P-value
At1g08430 0.01 ± 0.00 0.001 0.01 ± 0.00 0.000 0.01 ± 0.00 0.001 24.40 ± 13.8 0.009 7.41 ± 4.07 0.021 ATALMT1 (Al-activated malate transporter) At1g30270 0.13 ± 0.03 0.003 0.19 ± 0.02 0.001 1.02 ± 0.14 0.866 1.44 ± 0.09 0.009 1.42 ± 0.25 0.073 ATCIP K23 䠄CBL-interacting protein kinase 23) At1g51840 0.25 ± 0.06 0.010 0.40 ± 0.06 0.008 1.03 ± 0.15 0.765 1.31 ± 0.42 0.272 1.21 ± 0.30 0.305 similar to leucine-rich repeat protein kinase, putative At2g18480 0.06 ± 0.01 0.002 0.12 ± 0.09 0.031 1.08 ± 0.15 0.433 6.97 ± 1.12 0.002 1.89 ± 0.08 0.001 PLT3 (probable polyol transporter 3) At2g41380 0.04 ± 0.01 0.003 0.14 ± 0.08 0.025 1.11 ± 0.16 0.327 6.98 ± 3.70 0.021 2.28 ± 0.57 0.028 similar to embryo-abundant protein-related At5g02480 0.19 ± 0.03 0.003 0.31 ± 0.08 0.015 1.07 ± 0.15 0.491 2.02 ± 0.31 0.015 1.18 ± 0.07 0.040 similar to SLT1 (sodium䇲 and lithium䇲tolerant 1) At5g06860 0.05 ± 0.00 0.000 0.13 ± 0.06 0.017 1.17 ± 0.17 0.201 1.93 ± 1.46 0.232 1.00 ± 0.74 0.992 PGIP 1 (polygalacturonase inhibiting protein 1) At5g18170 0.08 ± 0.01 0.001 0.27 ± 0.11 0.030 1.26 ± 0.18 0.109 2.91 ± 0.39 0.005 1.19 ± 0.10 0.068 GDH1 (glutamate dehydrogenase 1) At5g19600 0.01 ± 0.00 0.002 0.29 ± 0.06 0.008 1.33 ± 0.19 0.074 1.50 ± 0.45 0.138 2.08 ± 0.37 0.019 SULTR3;5 (sulfate transporter 3;5)
At5g22890 0.06 ± 0.01 0.001 0.16 ± 0.08 0.023 1.07 ± 0.15 0.520 1.03 ± 0.20 0.809 1.15 ± 0.15 0.200 similar to STOP 1 (sensitive to proton rhizotoxicity 1) At5g38200 0.02 ± 0.00 0.001 0.10 ± 0.04 0.009 1.06 ± 0.15 0.552 4.16 ± 1.79 0.027 1.77 ± 0.19 0.012 similar to hydrolase
At5g66650 0.24 ± 0.03 0.003 0.19 ± 0.13 0.043 1.03 ± 0.15 0.766 9.13 ± 1.11 0.001 2.68 ± 1.17 0.055 similar to unknown protein At1g53480 0.02 ± 0.01 0.002 0.02 ± 0.01 0.007 0.01 ± 0.00 0.003 1.02 ± 0.02 0.341 1.10 ± 0.05 0.060 similar to unknown protein Downregulated in stop1 and not downregulated by Al; 24 genes
At1g35670 0.34 ± 0.02 0.001 0.76 ± 0.06 0.030 1.05 ± 0.15 0.598 1.35 ± 0.07 0.011 0.53 ± 0.08 0.020 ATCDP K2 (calcium-dependent protein kinase) At1g60610 0.26 ± 0.05 0.006 0.54 ± 0.05 0.008 0.95 ± 0.14 0.618 1.68 ± 0.23 0.023 1.16 ± 0.10 0.095 similar to protein binding / zinc ion binding At1g61560 0.26 ± 0.03 0.003 0.61 ± 0.09 0.029 0.99 ± 0.14 0.955 3.34 ± 0.75 0.011 2.65 ± 1.02 0.045 MLO6 (mildew resistance locus O 6) At1g72870 0.22 ± 0.09 0.021 0.59 ± 0.15 0.064 0.92 ± 0.13 0.406 1.29 ± 0.32 0.213 0.60 ± 0.05 0.008 putative disease resistance protein (TIR-NBS class) At1g77760 0.18 ± 0.05 0.008 0.88 ± 0.30 0.574 1.29 ± 0.19 0.088 1.66 ± 0.30 0.038 1.68 ± 0.13 0.007 NIA1 (nitrate reductase 1)
At2g01180 0.31 ± 0.03 0.003 0.59 ± 0.09 0.028 1.03 ± 0.15 0.779 2.13 ± 0.32 0.013 1.04 ± 0.09 0.525 ATP AP 1 (phosphatidic acid phosphatase 1) At2g16660 0.34 ± 0.01 0.001 0.53 ± 0.06 0.009 1.23 ± 0.18 0.126 2.62 ± 0.66 0.021 1.29 ± 0.43 0.310 similar to nodulin family protein At2g23150 0.31 ± 0.03 0.002 0.61 ± 0.13 0.054 1.04 ± 0.16 0.661 2.80 ± 0.55 0.012 1.27 ± 0.08 0.020 NRAMP 3 (manganese ion transporter) At2g28270 0.08 ± 0.03 0.006 0.41 ± 0.26 0.114 0.98 ± 0.23 0.873 2.52 ± 0.92 0.046 0.74 ± 0.34 0.357 similar to DC1 domain-containing protein
At2g39380 0.28 ± 0.05 0.005 0.83 ± 0.07 0.064 1.03 ± 0.15 0.753 1.41 ± 0.21 0.054 2.63 ± 0.22 0.003 ATEXO70H2 (exocyst subunit EXO70 family protein H2) At2g39510 0.27 ± 0.07 0.013 1.32 ± 1.12 0.579 1.35 ± 0.20 0.072 9.60 ± 7.83 0.032 1.97 ± 1.57 0.235 similar to nodulin MtN21 family protein
At2g43590 0.25 ± 0.10 0.025 1.09 ± 0.50 0.754 0.84 ± 0.15 0.157 3.20 ± 0.63 0.009 2.22 ± 2.00 0.214 putative chitinase
At2g45220 0.26 ± 0.04 0.005 0.69 ± 0.02 0.003 1.13 ± 0.29 0.458 1.98 ± 0.26 0.012 0.43 ± 0.11 0.026 similar to pectinesterase family protein At3g05400 0.19 ± 0.04 0.004 0.63 ± 0.15 0.076 1.20 ± 0.17 0.158 2.98 ± 1.04 0.031 1.07 ± 0.09 0.293 SUGTL5 (sugar transporter ERD6-like 12) At3g28290 0.13 ± 0.03 0.004 0.46 ± 0.16 0.055 0.87 ± 0.12 0.237 1.51 ± 0.13 0.014 0.93 ± 0.28 0.713 similar to AT14A
At4g13420 0.03 ± 0.00 0.000 2.21 ± 1.35 0.135 0.84 ± 0.12 0.165 7.54 ± 4.39 0.023 6.86 ± 1.12 0.002 HAK5 (high affinity K+ transporter 5) At4g16563 0.09 ± 0.07 0.026 0.22 ± 0.27 0.113 1.13 ± 0.28 0.449 1.22 ± 0.31 0.307 0.35 ± 0.04 0.004 similar to aspartyl protease family protein At4g30270 0.28 ± 0.06 0.010 0.84 ± 0.63 0.693 0.91 ± 0.15 0.407 12.56 ± 5.39 0.009 2.05 ± 0.76 0.074 MERI5B (endo xyloglucan transferase) At4g38470 0.29 ± 0.05 0.007 0.80 ± 0.22 0.297 1.17 ± 0.17 0.200 1.13 ± 0.20 0.343 1.15 ± 0.17 0.245 similar to protein kinase family protein At5g07440 0.29 ± 0.05 0.005 0.54 ± 0.15 0.060 1.16 ± 0.17 0.208 2.55 ± 0.16 0.001 1.23 ± 0.17 0.123 GDH2 (glutamate dehydrogenase 2) At5g11670 0.26 ± 0.07 0.012 0.67 ± 0.06 0.017 1.02 ± 0.14 0.834 3.62 ± 0.78 0.009 1.53 ± 0.15 0.018 ATNADP -ME2 (malic enzyme 2) At5g17860 0.31 ± 0.03 0.003 0.98 ± 0.02 0.208 0.98 ± 0.17 0.837 1.28 ± 0.06 0.012 1.02 ± 0.06 0.681 CAX7 (calcium exchanger 7)
At5g47560 0.12 ± 0.02 0.002 0.75 ± 0.15 0.129 1.09 ± 0.19 0.423 1.05 ± 0.12 0.557 1.06 ± 0.12 0.457 ATTDT (tonoplast malate/fumarate transporter) At5g66800 0.33 ± 0.03 0.003 0.58 ± 0.06 0.011 0.71 ± 0.11 0.057 1.04 ± 0.05 0.248 0.91 ± 0.06 0.124 similar to unknown protein
Downregulated in stop1 and not downregulated by pH; 4 genes
At1g62280 0.97 ± 0.62 0.936 0.47 ± 0.01 0.001 0.84 ± 0.15 0.161 0.33 ± 0.30 0.133 1.06 ± 0.51 0.858 SLAH1 (SLAC1 homologue 1) At1g75840 0.58 ± 0.04 0.005 0.47 ± 0.03 0.002 0.99 ± 0.15 0.879 0.94 ± 0.14 0.570 1.03 ± 0.06 0.460 ATROP 4 (rho-like gtp binding protein 4) At3g12750 0.60 ± 0.05 0.010 0.46 ± 0.09 0.020 1.42 ± 0.21 0.053 0.55 ± 0.04 0.004 1.62 ± 0.13 0.009 ZIP1 (zinc transporter 1)
At5g24780 0.65 ± 0.57 0.431 0.43 ± 0.01 0.000 2.21 ± 0.42 0.004 1.06 ± 1.02 0.918 4.00 ± 1.69 0.027 VSP1 (vegetative storage protein1)
T hree biologically independent replications of competitive microarray analyses were carried out as follows: stop1 mutant versus the wild type in Al (10 PM, 24 h) or low pH (pH 4.5, 24 h); AtALMT1 -KO versus the wild type in Al (10PM, 24 h); control (pH 5.0, no Al, 24 h) versus Al (10 PM, pH 5.0, 24 h) or low pH (pH 4.5, 24 h) in the wild type. FC 㼼 SD values are shown, as are P values from t tests for significant differences from FC = 1.00.
Downregulated in stop1 and not downregulated by Al and low pH; 12(+1) genes
Arabidopsis Genome Initiative Code
Table 3. List of repressed genes in the stop1 mutant among up-regulated or stable genes in the wild type under Al and low-pH conditions
Al stop1 -MT low pH stop1 -MT Al AtALMT -KO Al low pH FC (stop1 Mutant or AtALMT 1-KO/Wild T ype) FC in the Wild T ype with Al or Low-pH
T reatments (T reatment/Control)
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࣏࣮ࣜ࢜ࣝࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡢ PLT3ࠊAKT1 άᛶࢆㄪ⠇ࡍࡿ CBL-interacting
protein kinase 23 (CIPK23)ࠊNa+ / K+ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࢆㄪ⠇ࡍࡿSLT1 (sodium and lithium tolerant 1)ࡢ࣍ࣔࣟࢢ࡞ࠊࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡸ࢜ࣥ㍺㏦ࡢㄪ⠇ࢱ ࣥࣃࢡ࡞ࡀ㑅ᢤ㑇ఏᏊࡢࢢ࣮ࣝࣉぢࡽࢀࡓࠋࡉࡽࠊ❅⣲௦ㅰࡢ㔜せ࡞㑇ఏᏊ
ࡢ1 ࡘ࡛࠶ࡿ glutamate dehydrogenase 1 (GDH1)ࡸࠊ⏕≀ⓗ࡞ࢫࢺࣞࢫࡢ㜵ᚚࢩ
ࢫࢸ࣒㛵ࡍࡿ࠸ࢃࢀ࡚࠸ࡿpolygalacturonase inhibiting protein 1 (PGIP1)
ࡶྠࡌࢢ࣮ࣝࣉᒓࡋ࡚࠸ࡓࠋ
ࡑࡢࡢ Al ࡲࡓࡣప pH ࡛㌿ᢚไࡉࢀࡿ 2 ࡘࡢࢢ࣮ࣝࣉ࡛ࡣࠊ๓㏙ࡢ㑇ఏ
Ꮚ㛵㐃ࡍࡿᶵ⬟ࢆᣢࡘࡶࡢࡀ࠸ࡃࡘྵࡲࢀ࡚࠸ࡓ (Table 3)ࠋせ࡞࣒࢝ࣜ࢘
ࢺࣛࣥࢫ࣏࣮ࢱ࣮ࡢ 1 ࡘ࡛࠶ࡿ HAK5ࠊ❅⣲௦ㅰࡢせ࡞㓝⣲࡛࠶ࡿ NIA1
(nitrate reductase)ࡸ GDH2 ࡀ Al ฎ⌮ࡢࢢ࣮ࣝࣉྵࡲࢀ࡚࠸ࡓࠋALMT1 ␗ ࡞ ࡿ ࢱ ࣉ ࡢ ࣜ ࣥ ࢦ 㓟 ࢺ ࣛ ࣥ ࢫ ࣏ ࣮ ࢱ ࣮ ࠊAtTDT (vacuolar malate/Na+ co-transporter)ࡸࠊSLAC1 (slow anion channel 1)ࡢ࣍ࣔࣟࢢࠊࣜࣥࢦ㓟㓝⣲ࡢ ME2 (malic enzyme) ࡀ Al ฎ⌮ (AtTDTࠊME2)ࡸప pH (SLAC1 ࣍ࣔࣟࢢ)ฎ⌮
ࡢࢢ࣮ࣝࣉࡑࢀࡒࢀྵࡲࢀ࡚࠸ࡓࠋࡇࢀࡽࡢࢹ࣮ࢱࡣSTOP1 ࡢኚ␗ࡀ࢜ࣥࡢ ᜏᖖᛶࠊࣜࣥࢦ㓟ᨺฟࠊstop1 ኚ␗యࡢ௦ㅰࢆኚࡉࡏࡓࡇࢆ♧ࡋ࡚࠸ࡿࠋ sstop1 ኚ␗య࠾ࡅࡿ ALS3 ࡢ㌿ᢚไ పpH ឤཷᛶࡍࡿ㔜せ࡞㑇ఏᏊࡣ࠸ࡲࡔ᫂ࡽ࡞ࡗ࡚࠸࡞࠸ࡀࠊAl ⪏ᛶ ᛂ⟅ࡍࡿ࠸ࡃࡘࡢ㑇ఏᏊࡣࠊࢩࣟࢾࢼࢬࢼ࡛᫂ࡽ࡞ࡗ࡚࠸ࡿࠋ๓㏙ࡢࢹ࣮ ࢱᩚ⌮࡛ࡣࠊstop1 ኚ␗య࠾࠸࡚ࠊ᭷ព㌿ᢚไࡉࢀࡓ㑇ఏᏊࠊࡲࡓࡣ FC ್ ࡀୗ 1%ࡢ㑇ఏᏊࡢࡳ㑅ᢤࡋࡓࠋࡋࡋࠊࡶࡋ Al ⪏ᛶ㔜せ࡞㑇ఏᏊ࡞ࡽࡤࠊ ẚ㍑ⓗ㌿ᢚไࡉࢀ࡞࠸㑇ఏᏊࡀ stop1 ኚ␗యࡢ㉸ឤཷᛶࡢቑᐤࡋ࡚࠸ࡿ ࡶࡋࢀ࡞࠸ࠋࡇࢀࢆࠊྠࡌ࣐ࢡࣟࣞࡢFC (stop1 ኚ␗య / 㔝⏕ᆺ) ࢹ࣮ࢱ
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ࢆ⏝ࡋ࡚ࠊࡍ࡛▱ࡽࢀ࡚࠸ࡿ Al ⪏ᛶ㑇ఏᏊࡘ࠸࡚ㄪࡓࠋALS1 ALS3
ࡣኚ␗యゎᯒ࡛≉ᐃࡉࢀ (Larsen et al., 2005)ࠊWAK1 (cell wall-associated
kinase 1 (Sivaguru et al., 2003); At1g21250) ࠊ AtBCB (Arabidopsis blue copper-binding protein (Ezaki et al., 2004); At5g20230)ࠊAtCS (Arabidopsis citrate synthase (Koyama et al., 1999))ࡣ⤌యࡢゎᯒࡼࡗ࡚≉ᐃࡉࢀࡓࠋࡇࢀ
ࡽࡢ㑇ఏᏊࡢ୰࡛ࠊAl ឤཷᛶࡢჾᐁࡽ Al ࢆศ㓄ࡍࡿ㑇ఏᏊᒓࡍࡿ ALS3
(At2g37330)ࡔࡅࡀstop1 ኚ␗య࡛㌿ᢚไࡉࢀࠊFC ࡣ 0.33 ࡛࠶ࡗࡓ (Fig.3)ࠋ
ALS3 ࡢ㌿ࣞ࣋ࣝࢆᐃ㔞 RT-PCR ࡛ㄪࡓࡇࢁࠊstop1 ኚ␗య STOP1-KO
࡛ࡣ㌿ᢚไࡉࢀࠊCaMV35S::STOP1 ࢆstop1 ኚ␗య┦⿵ࡋࡓ࡛ࣛࣥࡣ㌿
ࣞ࣋ࣝࡣᅇࡋ࡚࠸ࡓ(Fig. 4a)ࠋࡇࡢ⤖ᯝࡣࠊᑡ࡞ࡃࡶࢩࣟࢾࢼࢬࢼ࡛ࡣ
STOP1 ࡀ AtALMT1 ࡸ ALS3 ࡢࡼ࠺࡞ Al ⪏ᛶ㔜せ࡞㑇ఏᏊࡢࢢ࣮ࣝࣉࢆㄪ⠇ ࡋ࡚࠸ࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋ
stop1 ኚ␗యࡢ Al ㉸ឤཷᛶ㛵ࡍࡿ Al ⪏ᛶ㑇ఏᏊ⩌ࡢ㌿ᢚไࢆホ౯ࡍࡿࡓ
ࡵࠊKO ኚ␗యࡢ⏕⫱ࢆẚ㍑ࡋࡓࠋ௨๓ሗ࿌ࡉࢀࡓࡼ࠺ (Iuchi et al., 2007)ࠊ
STOP1-KO ࡣప pH ឤཷᛶ࡛࠶ࡿࡀࠊAtALMT1-KO ࡣឤཷᛶ࡛ࡣ࡞࠸(Fig. 4b, 4c; Kobayashi et al., 2007)ࠋAl ࡢධࡗ࡚࠸࡞࠸ pH 5.5 ࡢࢥࣥࢺ࣮ࣟࣝ⁐ᾮ࡛ࡣࠊ
ࡍ࡚ࡢ KO ኚ␗యࡀ㔝⏕ᆺྠᵝᡂ㛗ࡋࡓࠋ୍᪉ࠊ㔝⏕ᆺࡢప pH ࢫࢺࣞࢫ (pH 5.5 ࡢ᰿ఙ㛗㜼ᐖẚ㍑)࡛ࡣࠊpH 5.0 ࡛ 30%⛬ᗘࡢࢃࡎ࡞㜼ᐖࢆ♧ࡋࠊࡉ ࡽpH 4.7 ࡛ࡣ⣙ 50㸣ࡢ㜼ᐖࢆ♧ࡋࡓࠋࡇࡢ᮲௳ୗ࡛ࠊALS3-KO ࡣ pH 5.0 pH 4.7 ࡛㔝⏕ᆺࡼࡾࡶࢃࡎⰋ࠸ᡂ㛗࡛࠶ࡗࡓࡇࡽࠊstop1 ኚ␗యࡢ ALS3 ࡢ㌿ᢚไࡣࠊపpH ࡢ㉸ឤཷᛶࡣ㛵㐃ࡀ࡞࠸ࡇࡀ♧ࡉࢀࡓࠋࡇࢀࡽࡢ KO ኚ ␗యࡢAl ឤཷᛶࢆホ౯ࡍࡿࡓࡵࠊప pH ࡢ᰿ẘᛶຠᯝࡀ᭱ᑠࠊࡘẚ㍑ⓗ㧗࠸ pH
᮲௳࡛ᐇ㦂ࢆ⾜ࡗࡓࠋ㐣ཤQTL (quantitative trait locus)ゎᯒ࡛ pH 5.0 ࢆ⏝
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Figure 3. Fold change of gene expression in microarray, whose gene is associated with Al tolerance as follows; AtALMT1 (Hoekenga et al., 2006), ALS1 (Larsen et al., 2007), ALS3 (Larsen et al., 2005), AtBCB (Ezaki et al., 2000), WAK1 (Sivaguru et al., 2003), AtCS (Koyama et al., 2000). Biologically independent 3 replication of competitive microarray analyses were carried out as follows; stop1-mutant versus wild type in Al (10 M, 24 h; light gray) and low pH (pH 4.5, 24 h; dark gray). Fold change and whose s SD are shown.
0.0 0.5 1.0 1.5 2.0 AtA LMT1 ALS1 ALS3 AtBC B WAK1 AtC S Fol d change
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Figure 4. Repression of ALS3 in the stop1 mutant and comparison of growth among T-DNA insertion mutants. (a) ALS3 expression in the wild type (WT), the stop1 mutant, STOP1-KO, and a complemented line of the stop1 mutant carrying CaMV35S::STOP1 (stop1-comp), which were grown for 10 d in normal conditions and exposed to Al (10 M, pH 5.0) for 24 h. ALS3 expression was normalized by UBQ1 expression and compared with that of the wild type. (b) and (c), Growth response of ALS3-KO compared with that of STOP1-KO and AtALMT1-KO with Al and H+ rhizotoxicities. White bar in B = 1 cm. Different letters indicate significant differences by LSD test (P = 0.05).
